Methods for processing nucleic acid samples

ABSTRACT

The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and/or DNA molecules and cDNA and/or DNA libraries using modified reverse transcriptases.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/US2017/061197, filed Nov. 10, 2017, which claims the benefit of U.S.Provisional Application No. 62/421,028, filed Nov. 11, 2016 and U.S.Provisional Application No. 62/477,211, filed Mar. 27, 2017, each ofwhich is entirely incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 18, 2018, isnamed 51160_701_303_SL.txt and is 732,332 bytes in size.

BACKGROUND

A common technique used to study gene expression in living cells is toproduce complementary deoxyribonucleic acid (cDNA) from a ribonucleicacid (RNA) molecule. This technique provides a means to study RNA fromliving cells which avoids the direct analysis of inherently unstableRNA. As a first step in cDNA synthesis, the RNA molecules from anorganism are isolated from an extract of cells or tissues of theorganism. After messenger RNA (mRNA) isolation, using methods such asaffinity chromatography utilizing oligo dT (a short sequence ofdeoxy-thymidine nucleotides), oligonucleotide sequences are annealed tothe isolated mRNA molecules and enzymes with reverse transcriptaseactivity can be utilized to produce cDNA copies of the RNA sequence,utilizing the RNA/DNA primer as a template. Thus, reverse transcriptionof mRNA is a key step in many forms of gene expression analyses.Generally, mRNA is reverse transcribed into cDNA for subsequent analysisby primer extension or polymerase chain reaction.

Reverse transcriptase has both an RNA-directed DNA polymerase activityand a DNA-directed DNA polymerase activity. The reverse transcription ofRNA templates may require a primer sequence which is annealed to an RNAtemplate in order for DNA synthesis to be initiated from the 3′ OH ofthe primer. At room temperature, reverse transcriptase enzymes may allowformation of both perfectly matched as well as mismatched DNA/RNAhybrids. In some instances, a reverse transcriptase enzyme can producelarge amounts of non-specific cDNA products as a result of suchnon-specific priming events. The products of non-specific reversetranscription can interfere with subsequent cDNA analyses, such as cDNAsequencing, real-time polymerase chain reaction (PCR), and alkalineagarose gel electrophoresis, among others. Non-specific cDNA templatesproduced by non-specific reverse transcriptase activity can presentparticular difficulties in applications such as real-time PCR. Inparticular, such non-specific cDNA products can give rise to falsesignals which can complicate the analysis of real-time PCR signals andproducts. Thus, the reduction of non-specific reverse transcriptaseactivity may result in greater specificity of cDNA synthesis. Currently,there are no reliable and easy to use methods for improving thespecificity of reverse transcription. The present disclosure satisfiesthese and other needs.

Several approaches may be used for obtaining transcriptome data fromsingle cells. A pioneer approach used reverse transcriptase and oligo-dTprimers with a T7 phage RNA polymerase promoter sequence attached to the5′ end of the oligo-dT run. The resulting cDNA was transcribed intomultiple copies of RNA which were then converted back to cDNA (Phillips,et al., Methods 10(3):283-288 (1996)). This often truncates the cDNAmolecule, losing 5′ sequences of the original mRNA, especially forrelatively long transcripts, and requires multiple rounds of processingwhen starting with low quantity (LQ) of cells, further exacerbating cDNAtruncation. A recent modification (Hashimshony, et al., Cell Rep.2(3):666-673 (2012)) enables multiplex analyses, but this is still 3′end sequence biased. Other methods are based on PCR amplification ofcDNA (Liu, et al., Methods Enzymol. 303:45-55 (1999), Ozsolak, et al.,Genome Res. 20(4):519-525 (2010), Gonzalez, et al., PLoS ONE.5(12):e14418 (2010), Kanamori, et al., Genome Res. 21(7):1150-1159(2011), Islam, et al., Genome Res. 21(7):1160-1167 (2011), Tang, et al.,Nat. Methods. 6(5):377-382 (2009), Kurimoto, et al., Nucleic Acids Res.34(5):e42 (2006), Qiu S, et al., Front Genet. 3:124 (2012)).

These approaches, however, may yield biased representations of sequencesalong the mRNA, and fail to give complete sequences for mRNAs (e.g.,long mRNAs) because DNA templates (e.g., long DNA templates) arediscriminated against even when a long PCR reaction is used.

SUMMARY

The present disclosure provides methods of amplifying cDNA from RNAisolated from low quantities of cells and/or single cells. The presentdisclosure also provides methods of transcriptome analysis where cellsdo not go through stress (e.g. elevated temperature for cDNA analysis),thus maintaining the transcriptome profile.

In one embodiment, the present disclosure relates to a method forpreparing a complementary deoxyribonucleic acid (cDNA) moleculecomprising:

-   -   (a) annealing a primer to a template nucleic acid molecule,        thereby generating an annealed template nucleic acid molecule;        and    -   (b) mixing, in the presence of nucleotides,        -   i. said annealed template nucleic acid molecule;        -   ii. one or more acceptor nucleic acid molecules; and        -   iii. a modified reverse transcriptase, wherein said modified            reverse transcriptase generates a plurality of continuous            complementary deoxyribonucleic acid molecules by: i) reverse            transcribing a sequence of said annealed template nucleic            acid molecule; ii) migrating to an acceptor nucleic acid            molecule; and iii) reverse transcribing a sequence of said            acceptor nucleic acid molecule at a temperature of from            about 12° C. to about 42° C. with an error rate of at most            about 5%.

In some embodiments, the migrating is independent of sequence identitybetween the template and the acceptor nucleic acid molecule. In someembodiments, (a) and (b) are performed in a single vessel. In someembodiments, the method further comprises performing the method on aheterogeneous plurality of template nucleic acid molecules comprising aplurality of distinct ribonucleic acid (RNA) molecules. In someembodiments, the plurality of distinct ribonucleic acid moleculescomprise messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs(tRNAs), micro RNAs (miRNAs), and long non-coding RNAs (lncRNAs). Insome embodiments, the plurality of continuous complementarydeoxyribonucleic acid molecules is prepared in at most about 2 hours. Insome embodiments, the template nucleic acid molecule is selected fromthe group consisting of an artificially fragmented DNA template, anaturally fragmented DNA template, an artificially fragmentedribonucleic acid (RNA) template, a naturally fragmented ribonucleic acid(RNA) template, or a combination thereof.

In some embodiments, the modified reverse transcriptase amplifies saidannealed template nucleic acid molecule at a processivity of at leastabout 80% per base. In some embodiments, the modified reversetranscriptase comprises at least one improved enzyme property relativeto a wild type unmodified reverse transcriptase. In some embodiments, atleast one improved enzyme property is selected from the group consistingof: higher thermo-stability, higher specific activity, higherprocessivity, higher strand displacement, higher end-to-end templatejumping, higher affinity, and higher fidelity relative to said wild typeunmodified reverse transcriptase. In some embodiments, modified reversetranscriptase is an R2 reverse transcriptase. In some embodiments, themodified reverse transcriptase has at least about 90% identity to SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67, andcontains at least one substitution modification relative to SEQ ID NO:52.

In some embodiments, the method further comprises adding a tag to thetemplate nucleic acid molecule, thereby generating a plurality of taggedcontinuous complementary deoxyribonucleic acid molecules afterperforming (a) and (b). In some embodiments, the method furthercomprises sequencing the tagged plurality of continuous complementarydeoxyribonucleic acid molecules. In some embodiments, modified reversetranscriptase further comprises a tag. In some embodiments, the tag isselected from the group consisting of biotin, azido group, acetylenegroup, His-tag, calmodulin-tag, CBP, CYD, Strep II, FLAG-tag, HA-tag,Myc-tag, S-tag, SBP-tag, Softag-1, Softag-3, V5-tag, Xpress-tag,isopeptag, SpyTag B, HPC peptide tags, GST, MBP, biotin carboxyl carrierprotein, glutathione-S-transferase-tag, green fluorescent protein-tag,maltose binding protein-tag, Nus-tag, Strep-tag, and thioredoxin-tag.

In some embodiments, the method further comprises performing apolymerase chain reaction (PCR) amplification reaction, thereby formingone or more amplicons. In some embodiments, (a) and (b) and the PCRamplification reaction are performed in the same vessel. In someembodiments, the PCR amplification is performed at a temperaturesufficient to inactivate the reverse transcriptase.

In some embodiments, the one or more acceptor nucleic acid moleculescomprise a modified nucleotide that stops the reverse transcription bythe modified reverse transcriptase. In some embodiments, the methodfurther comprises obtaining a sample comprising one or more templatenucleic acid molecules from a subject and annealing one or more primersto said one or more template nucleic acid molecules. In someembodiments, the sample is a tissue sample. In some embodiments, thesample comprises one or more cell free nucleic acids and the methodfurther comprises performing the reaction of (a) and (b) in the samevessel.

In some embodiments, the method further comprises depleting at least oneribosomal RNA (rRNA) from the sample comprising one or more templatenucleic acid molecules prior to annealing one or more primers. In someembodiments, depleting the at least one ribosomal RNA (rRNA) compriseshybridization of an oligonucleotide to the rRNA. In some embodiments,depleting said at least one ribosomal RNA (rRNA) comprises anoligonucleotide probe-guided endonucleolitic cleavage of the rRNA. Insome embodiments, the one or more template nucleic acid moleculescomprise at least one transfer RNA (tRNA) and the method furthercomprises depleting the at least one tRNA from the one or more templatenucleic acid molecules prior to annealing one or more primers.

In some embodiments, the method is performed in the absence ofpurification of one or more annealed template nucleic acid molecules. Insome embodiments, the method further comprises purifying the mixturecomprising a plurality of continuous complementary deoxyribonucleic acidmolecule. In some embodiments, purifying comprises two purificationsteps. In some embodiments, the two purification steps comprise a nickeland a heparin affinity purification steps.

In some embodiments the method is performed on from about 0.1 nM toabout 100 nM of one or more template nucleic acid molecules. In someembodiments, the method further comprises annealing one or more randomprimer(s) to the template nucleic acid molecule. In some embodiments, aprimer hybridizes to one or more adapter sequence. In some embodiments,the template nucleic acid molecule is derived from a single cell.

In one embodiments, the present disclosure relates to a method forpreparing a complementary deoxyribonucleic acid molecule comprising:

-   -   (a) annealing one or more primers to an amount of template        nucleic acid molecules, thereby generating one or more annealed        template nucleic acid molecules; and    -   (b) mixing, in the presence of nucleotides,        -   i. said one or more annealed template nucleic acid            molecules;        -   ii. one or more acceptor nucleic acid molecules; and        -   iii. a modified reverse transcriptase, whereby said modified            reverse transcriptase generates a plurality of continuous            complementary deoxyribonucleic acid molecule by reverse            transcribing a sequence of an annealed template nucleic acid            molecule, migrating to an acceptor nucleic acid molecule,            and reverse transcribing a sequence of said acceptor nucleic            acid molecule without thermal cycling in a single reaction            vessel.

In some embodiments, the migrating is independent of sequence identitybetween the template and the acceptor nucleic acid molecule. In someembodiments, the plurality of continuous complementary deoxyribonucleicacid molecules are prepared in at most about 2 hours. In someembodiments, the amount of template nucleic acid molecules is selectedfrom the group consisting of an artificially fragmented DNA template, anaturally fragmented DNA template, an artificially fragmentedribonucleic acid (RNA) template, a naturally fragmented ribonucleic acid(RNA) template, or a combination thereof. In some embodiments, theamount of template nucleic acid molecules comprises one or more cellfree nucleic acids. In some embodiments, the modified reversetranscriptase comprises at least one improved enzyme property relativeto a wild type unmodified reverse transcriptase. In some embodiments,the improved enzyme property is selected from the group consisting of:higher thermo-stability, higher specific activity, higher processivity,higher strand displacement, higher end-to-end template jumping, higheraffinity, and higher fidelity relative to said wild type unmodifiedreverse transcriptase. In some embodiments, the modified reversetranscriptase is an R2 reverse transcriptase. In some embodiments, themodified reverse transcriptase has at least 90% identity to SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ IDNO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67 andcontains at least one substitution modification relative to SEQ ID NO:52.

In some embodiments, the method further comprises adding a tag to theamount of template nucleic acid molecules, thereby generating aplurality of tagged continuous complementary deoxyribonucleic acidmolecules after performing (a) and (b). In some embodiments, the methodfurther comprises sequencing the plurality of tagged continuouscomplementary deoxyribonucleic acid molecules. In some embodiments, themodified reverse transcriptase comprises a tag. In some embodiments, thetag is selected from the group consisting of biotin, azido group,acetylene group, His-tag, calmodulin-tag, CBP, CYD, Strep II, FLAG-tag,HA-tag, Myc-tag, S-tag, SBP-tag, Softag-1, Softag-3, V5-tag, Xpress-tag,isopeptag, SpyTag B, HPC peptide tags, GST, MBP, biotin carboxyl carrierprotein, glutathione-S-transferase-tag, green fluorescent protein-tag,maltose binding protein-tag, Nus-tag, Strep-tag, and thioredoxin-tag. Insome embodiments, the one or more acceptor nucleic acid moleculescomprises a modified nucleotide that stops the reverse transcription bysaid modified reverse transcriptase.

In some embodiments, the method further comprises obtaining a samplefrom a subject. In some embodiments, the sample comprises one or morecell free nucleic acids and the method further comprises performing thereaction of (a) and (b) in the same vessel. In some embodiments, thesample is a tissue sample. In some embodiments, the method furthercomprises depleting at least one ribosomal RNA (rRNA) from the one ormore template nucleic acid molecules prior to annealing one or moreprimers. In some embodiments, depleting said at least one ribosomal RNA(rRNA) comprises hybridization of an oligonucleotide to the rRNA. Insome embodiments, depleting the at least one ribosomal RNA (rRNA)comprises an oligonucleotide probe-guided endonucleolitic cleavage ofthe rRNA. In some embodiments, depleting at least one transfer RNA(tRNA) from the amount of template nucleic acid molecules is prior toannealing one or more primers.

In some embodiments, the method is performed in the absence ofpurification of one or more annealed template nucleic acid molecules. Insome embodiments the method further comprises purifying the mixturecomprising the plurality of continuous complementary deoxyribonucleicacid molecules. In some embodiments, purifying comprises twopurification steps. In some embodiments, the two purification stepscomprise a nickel and a heparin affinity purification steps. In someembodiments, the amount of template nucleic acid molecules is from about0.1 nM to about 100 nM of said one or more template nucleic acidmolecules. In some embodiments, the one or more primers comprise one ormore random primer(s). In some embodiments, the one or more primers ishybridized to one or more adapter sequence. In some embodiments, theamount of template nucleic acid molecules are derived from a singlecell.

In one embodiment, the present disclosure relates to a polypeptidehaving reverse transcriptase activity comprising an amino acid sequencethat has at least 90% identity to SEQ ID NO: 49, SEQ ID NO: 50, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO: 66, or SEQ ID NO: 67 and contains at least onesubstitution modification relative to SEQ ID NO: 52.

In one embodiment, the present disclosure relates to a purifiedpolypeptide comprising one or more domains from an R2 reversetranscriptase wherein said purified polypeptide generates one or morecopies of a complementary deoxyribonucleic acid molecule from a templatenucleic acid molecule with an error rate of at most about 5%.

In some embodiments, the polypeptide reverse transcribes a templatenucleic acid molecule at a processivity of at least about 80% per base.In some embodiments, the polypeptide reverse transcribes a templatenucleic acid molecule at a temperature of from about 12° C. to about 42°C. In some embodiments, the polypeptide comprises an amino acid sequenceas set forth in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66,or SEQ ID NO: 67.

In one embodiment, the present disclosure relates to a non-naturallyoccurring enzyme comprising one or more domains from an R2 reversetranscriptase wherein the non-naturally occurring enzyme generates acomplementary deoxyribonucleic acid product in a time period of lessthan about three hours and at a performance index greater than 1.0 forat least one enzyme property selected from the group consisting ofimproved stability, specific activity, protein expression, purification,processivity, strand displacement, template jumping, increased DNA/RNAaffinity, and fidelity, as compared to a purified enzyme of SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ IDNO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67.

In some embodiments, an R2 reverse transcriptase and/or a non-naturallyoccurring enzyme and/or a polypeptide can have at least one mutationand/or modification. In some embodiments, an R2 and/or a non-naturallyoccurring enzyme and/or a polypeptide comprises a C952S, and/or C956S,and/or C952S, C956S (double mutant), and/or C969S, and/or H970Y, and/orR979Q, and/or R976Q, and/or R1071S, and/or R328A, and/or R329A, and/orQ336A, and/or R328A, R329A, Q336A (triple mutant), and/or G426A, and/orD428A, and/or G426A, D428A (double mutant) mutation, and/or anycombination thereof.

In one embodiment, the present disclosure relates to a methodcomprising:

-   -   (a) adding a nuclease complex to a plurality of double-stranded        nucleic acid molecules, wherein said nuclease complex comprises:        -   i. a Cas9 nuclease or a functional variant thereof; and        -   ii. at least one synthetic guide oligonucleotide, wherein            said synthetic guide oligonucleotide is complementary to a            ribosomal ribonucleic acid (rRNA) or transfer ribonucleic            acid (tRNA) region in at least one double-stranded nucleic            acid molecule in said plurality of double-stranded nucleic            acid molecules;    -   (b) permitting said complex to cleave said rRNA or tRNA region        of at least one double-stranded nucleic acid molecule, thereby        providing at least one cleaved double-stranded nucleic acid        molecule, and    -   (c) subjecting said at least one cleaved double-stranded nucleic        acid molecule or derivative thereof to nucleic acid sequencing,        thereby yielding a nucleic acid sequence of said at least one        double-stranded nucleic acid molecule lacking said rRNA or tRNA        region.

In some embodiments, the synthetic guide oligonucleotide iscomplementary to a ribosomal ribonucleic acid (rRNA). In someembodiments, the synthetic guide oligonucleotide is complementary to atransfer ribonucleic acid (tRNA). In some embodiments, the method doesnot require denaturation of the plurality of double-stranded moleculescomprising sequences derived from rRNAs and tRNAs prior to sequencing.In some embodiments, the plurality of double-stranded nucleic acidmolecules comprise a cDNA.

In some embodiments, the method does not require denaturation of theplurality of double-stranded molecules comprising sequences derived fromrRNAs and/or tRNAs. In some embodiments, the plurality ofdouble-stranded nucleic acid molecules comprises sequences derived froman rRNA and/or a tRNA. In some embodiments, the double-stranded nucleicacid molecules comprises cDNA. In some embodiments, the pre-determinedregion is a region of an rRNA. In some embodiments, the pre-determinedregion is a region of a tRNA.

In one embodiment, the present disclosure relates to a method forpreparing a concatemer of nucleic acid molecules comprising:

-   -   (a) processing ends of a plurality of double-stranded nucleic        acid molecules;    -   (b) adding a first plurality of adaptor molecules to said        plurality of double stranded nucleic acid molecules, wherein        said first plurality of adaptor molecules comprise one or more        overhang sequences, wherein at least two overhang sequences are        complementary to each other, thereby providing a first plurality        of adaptor connected double-stranded nucleic acid molecules;    -   (c) adding a polymerizing enzyme to said first plurality of        adaptor connected double-stranded nucleic acid molecules in the        absence of a primer, whereby said polymerizing enzyme forms a        first set of adaptor connected double-stranded nucleic acid        concatemers by joining two or more adaptor connected        double-stranded nucleic acid molecules by said one or more        overhang sequences;    -   (d) adding a second plurality of adaptor molecules to said first        set, wherein said second plurality of adaptor molecules comprise        one or more overhang sequences, wherein at least two overhang        sequences are complementary to each other, thereby providing a        second set of adaptor connected double-stranded nucleic acid        molecules;    -   (e) repeating (a)-(c) with a set of adaptor molecules to yield a        concatemer comprising a predetermined average length.

In some embodiments, the processing of (a) comprises end repair. In someembodiments, the polymerizing enzyme is a reverse transcriptase. In someembodiments, the reverse transcriptase is an R2 reverse transcriptase ora functional variant thereof. In some embodiments, the first pluralityof adaptor molecules, the second plurality of adaptor molecules, or bothcomprise a unique molecular identifier sequence (UMI). In someembodiments, the polymerizing enzyme joins two or more adaptor connecteddouble-stranded nucleic acid molecules in a PCR or isothermalamplification reaction. In some embodiments, the first plurality ofadaptor molecules, the second plurality of adaptor molecules, or bothcomprise at least one modified nucleotide

In some embodiments, the adaptor comprises a unique molecular identifiersequence (UMI). In some embodiments, the amplifying is performed by PCRor isothermal amplification. In some embodiments, the adaptor comprisesat least one modified nucleotide.

Advantages of the present disclosure include, but is not limited to:efficient and simple method for cDNA and nucleic acid librarypreparation (e.g., single cell and/or bulk library preparation) that iscompatible with various sequence technologies; high quality librarypreparation that can be used for single cell nucleic acid (e.g., RNA)sequencing and bulk nucleic acid (e.g., RNA) sequencing; modifiedreverse transcriptase enzymes with improved enzyme property; highconversion (e.g., efficiency and/or fidelity) of a nucleic acid sample(e.g., RNA, and/or mRNA, and/or DNA) to nucleic acid (e.g., cDNA)library; low non-specific products yield; and transcriptome analysiswhere cells don't go through stress (e.g., no need for elevatedtemperature for cDNA synthesis) because nucleic acid synthesis may beperformed at ambient temperatures (e.g., 30° C.).

Advantages of the present disclosure also include the ability to producea nucleic acid (e.g., cDNA) library using random or multi-priming and/ora library from fragmented/degraded nucleic acid molecule(s) (e.g., RNAand DNA), even at low amounts (e.g., 500 femtomolar) offragmented/degraded nucleic acid molecule(s). Advantages of the presentdisclosure also include the ability of the disclosed methods (e.g., byusing a modified reverse transcriptase) to amplify a fragment (e.g.,amplification of the full fragment) and generate multiple copies of thefull fragment for sequencing. Current available methods may cause afragment to be amplified at a random location and may only amplifysections of the fragment, thus sequencing and identification of thefragment(s) according to current methods is not available at lowamounts. Advantages of the present disclosure also include amplificationvia a single step or via a two step amplification protocol, thusincreasing specificity and efficiency. Current methods include multiplestep amplification that can result in low yield, low efficiency, and/orlow specificity. Another advantage of the methods of the presentdisclosure is the capability to prime using a piece of nucleic acidmolecule (e.g., RNA) that does not have to be complementary to atemplate. Another advantage of the present disclosure is the ability ofthe modified enzymes to template jump at room temperature and/or attemperatures as low as about 30° C. Another advantage of the presentdisclosure is the ability to prepare a library from a sample in lessthan three steps (FIG. 1) and/or less than 4 hours. This advantage isparticular important for clinical research and testing and the medicalfield. Another advantage of the present disclosure is the ability of themethods disclosed herein (e.g., by using a modified reversetranscriptase) to improve template jumping, processivity, stranddisplacement properties, enzyme activity, and/or fidelity.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications, and NCBI accessionnumbers mentioned in this specification are herein incorporated byreference to the same extent as if each individual publication, patent,patent application, or NCBI accession number was specifically andindividually indicated to be incorporated by reference. To the extentpublications and patents, patent applications, or NCBI accession numbersincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 illustrates a difference between preparing a complementarydeoxyribonucleic acid (cDNA) library between a traditional method andthe method of the present disclosure. FIG. 1 also illustrates adifference between preparing a sample for sequencing from a liquidbiopsy sample based on a traditional method and the method of thepresent disclosure. The traditional method involves a protocol thatrequires about 1 to 2 days and more than 4-5 hours of hands-on time,while the method of the present disclosure involves a protocol thatrequires less than about 2 hours and less than about 30 minutes ofhands-on time;

FIG. 2 illustrates a workflow for constructing a library based on themethods of the present disclosure;

FIG. 3 illustrates a workflow for constructing a library;

FIG. 4 illustrates a workflow using random primers for constructing alibrary;

FIG. 5A illustrates a workflow using fragmented or degraded ribonucleicacid (RNA) or deoxyribonucleic acid (DNA) with RNA priming forconstructing a library;

FIG. 5B illustrates an example of a workflow using fragmented ordegraded RNA or DNA with RNA priming for constructing a library—methodwith specific primer;

FIG. 6A illustrates a workflow using fragmented or degraded RNA or DNAwith a donor complex for constructing a library;

FIG. 6B illustrates an example of a workflow using fragmented ordegraded RNA or DNA with a donor complex for constructing a library—withspecific primer;

FIG. 7 illustrates a schematic representation of an N-terminal and aC-terminal truncation of an R2 reverse transcriptase;

FIG. 8 illustrates a sequence analysis with selected non-long terminalrepeat (LTR) retrotransposon and an example of a site of N-truncationupstream two conservative regions (region −1 and region 0);

FIG. 9 illustrates a sequence analysis with selected non-LTRretrotransposon and an example of a site of C-terminal truncationdownstream two conservative motifs 8* and 9*;

FIG. 10 illustrates a gel showing purified wild type R2 reversetranscriptase and N-terminal truncated R2 reverse transcriptase;

FIG. 11 illustrates a polyacrylamide gel electrophoresis (PAGE) gelshowing activity and template jumping properties of an R2 enzyme and ofmoloney murine leukemia virus (MMLV) reverse transcriptase by usingsynthetic RNA;

FIG. 12A illustrates a workflow for sequencing library preparation;

FIG. 12B illustrates real-time PCR data of library preparation based on1-pot (e.g., single vessel) reactions;

FIG. 12C illustrates a gel showing amplicon;

FIG. 13 illustrates 1-pot (e.g., single vessel) RNA library preparationusing different template amounts;

FIG. 14 illustrates 1-pot (e.g., single vessel) RNA library preparationwith different template lengths;

FIG. 15 illustrates that enzyme activity and template jumping isdependent on the concentration of sodium chloride (NaCl);

FIG. 16A illustrates enzyme activity after nickel and/or heparinaffinity purification visualized based on fluorescently labeled primer(fluorescein);

FIG. 16B illustrates template jumping properties after nickel and/orheparin affinity purification visualized based on Sybr gold staining;

FIG. 17 illustrates R2 enzyme activity and template jumping in thepresence of DNA template (lane 1: no enzyme control; lane 2: 0.023 μg/μlenzyme in the presence of DNA template);

FIG. 18A illustrates a workflow of a method of the present disclosurefor sequencing a library preparation from liquid biopsy;

FIG. 18B illustrates that a sample DNA fragment was captured by an R2enzyme with both an RNA priming approach and an RNA donor approach;

FIG. 18C illustrates that a sample DNA fragment (at variousconcentrations) was captured by an R2 enzyme using an RNA primingapproach;

FIG. 18D illustrates that a sample DNA fragment (at variousconcentrations) was captured by an R2 enzyme using an RNA donorapproach;

FIG. 19 illustrates that a sample DNA fragment was captured using an RNAdonor approach at a concentration as low as 500 femtomolar;

FIG. 20 illustrates that the method of the present disclosure can beused for liquid biopsy application. FIG. 20 discloses that the methodsdescribed herein show high sensitivity compared to current availabletechnology (e.g., the methods disclosed herein can be used for very lowDNA amounts, as low as 0.3 pg, which is about a 100-1000 fold highersensitivity than current available methods). Data also shows thepotential for applications involving DNA amounts that are even lowerthan 0.3 pg (e.g., a few orders of magnitude lower);

FIG. 21 illustrates a method of the present disclosure that takesadvantage of detecting mutations on both RNA (e.g., exosomal RNA) andcell-free DNA. This method can be used to detect rare mutations or lowfrequency mutations (e.g., some mutated alleles can occur at less than 1copy per mL of plasma) by increasing the detection sensitivity bycombining RNA and cell-free DNA. This method can be used, for example,to test analytes isolated from body fluids (e.g., blood). Body fluids,such as blood plasma, may contain different cell-free sources of nucleicacids. Such cell-free sources can be circulating cell-free DNA (e.g.,double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA)) and RNA(e.g., extracellular RNA (exRNA) and RNA from exosomes). Cell-free DNAcan be present at various stages of fragmentation and/or degradation(e.g., different lengths). Extracellular RNA can include, but is notlimited to, messenger RNA (mRNA), transfer RNA (tRNA), microRNA (miRNA),small interfering RNA (siRNA), and long non-coding RNA (lncRNA). FIG. 21also illustrates a method of the present disclosure that discriminatelytag cell-free DNA and RNA present in the same, or different, tube (e.g.,same PCR tube). Tagging facilitates post-sequencing analysis by allowingdiscrimination between sequences that originated from DNA and RNAtemplates. FIG. 21 shows that two different analytes, DNA and RNA, werepolytailed in the presence of Terminal Deoxynucleotidyl Transferase(TdT), Poly A Polymerase, and specific nucleotide substrates dCTP (oralternatively dGTP or dTTP) and ATP. In short, a reaction containingboth RNA and DNA (dsDNA and ssDNA) was mixed and incubated with poly Apolymerase, TdT, dCTP, and ATP (FIG. 21). Poly A polymerasepreferentially extends RNA using the preferred substrate ATP while TdTpreferentially extends DNA using the preferred deoxy substrate dCTP. Ingeneral, the reaction can be performed with both enzymes (e.g., poly Apolymerase and TdT) at the same time, or alternatively, it can beperformed sequentially with one enzyme at a time;

FIG. 22A illustrates that streptavidin-immobilized oligonucleotides canbe used for improving the efficiency of specific template capture andtemplate jumping capabilities. FIG. 22A shows thatstreptavidin-immobilized oligonucleotides bind to specific DNA and/orRNA template(s). In this case, the streptavidin-immobilizedoligonucleotides are bound to magnetic beads. Once the specific DNAand/or RNA template is bound to the streptavidin-magnetic bead complex,the template can be enriched. The oligonucleotide can be used as aprimer and the template can be transcribed in the presence of an enzyme(e.g., R2 enzyme);

FIG. 22B illustrates that a complex comprising of streptavidin bound toboth an oligonucleotide primer and an oligonucleotide acceptor iscapable of binding to specific DNA and/or RNA template(s). In short, thespecific template binds to the oligonucleotide primer, which can then beextended in the presence of an enzyme (e.g., R2 enzyme). The primerextension then undergoes template jumping due to the close proximitybetween the acceptor oligonucleotide and the specific template;

FIG. 22C illustrates that a complex comprising of streptavidin bound toan oligonucleotide primer, an oligonucleotide acceptor, and a magneticbead is capable of binding to specific DNA and/or RNA template(s). Inthis case, the specific template is first enriched with magnetic beads.The template is then copied in the presence of an enzyme (e.g., R2enzyme) and the extended sequence can further undergo template jumping;

FIGS. 23A and 23B illustrate template concatemerization. Some sequencingtechnologies have a long sequencing read-length (˜500 bp to ˜50000 bp)while others have a short sequencing read-length (˜50 bp to ˜250 bp).Most of the isolated cell-free DNA and RNA from body fluids are shortfragments (˜20 bp to ˜200 bp). The method shown in FIGS. 23A and 23B isparticularly suitable for sequencing technologies that have a longsequencing read-length because the method is capable of forming longtemplate concatemers. FIG. 23A illustrates a method of concatemerizingseveral templates separated by signaling sequences. In this method,short dsDNA fragments are converted to a long concatemer separated bysignaling sequences. FIG. 23B shows a final product of aconcatemerization that includes specific adaptors on both ends. Theadaptor design incorporates unique molecular identifier sequences (UMI)that allow one to trace the tagged molecule and also help reduce errorsduring data analysis. In short, dsDNA fragments are ligated with two ormore adaptors. The ligated fragments are then extended using PCR withoutprimers (alternatively isothermal amplification). The concatemer lengthor the number of attached templates can be determined, for example, bytagging the adaptors with modified nucleotides (e.g., by introducingmethylated nucleotides or by inserting dUTP). The length of theconcatemer can be regulated based on the ratio betweenmodified/unmodified adaptors. The adaptor sequences can serve as ahomology priming location (annealed to the homology spot ssDNA fragmentsserve as template and primer). The reaction in the PCR undergoes aselected number of cycles (the more cycles, the longer the concatemer)or time (isothermal amplification). The reaction is then stopped and thelong dsDNA concatemers are ligated with two unique dsDNA adaptors. Seealso, Example 13;

FIG. 24A illustrates a workflow of template concatemerization of thepresent disclosure;

FIG. 24B illustrates a gel showing the concatemerization of a 200 bp DNAfragment;

FIG. 25 illustrates a schematic reaction in the presence of a reversetranscriptase and a second enzyme or enzymatic activity (i.e., companionenzyme), such as an ssDNA 3′ to 5′ exonuclease or a polymerase withediting activity (e.g., 3′ to 5′ exonuclease). Examples of a companionenzyme include, but are not limited to, T4 DNA polymerase, exonucleaseI, and exonuclease T. One function or purpose of the companion enzyme isto remove the excess of free unused extension primer. Free primer maycontribute to unwanted products (e.g., free primer may serve as ajumping acceptor or it may be used as a nonspecific primer). FIG. 25shows a reaction scheme which starts with the annealing of a primer toan RNA template (the primer can be annealed to a specific sequence, orto a polyA tail, or to a product of poly-tailing of the 3′ end). Thereaction is then mixed with an enzyme (e.g., R2 enzyme), a polymerasewith editing activity (e.g., 3′ to 5′ exonuclease), and an acceptortemplate (e.g., acceptor template with a protected 3′ end). The acceptortemplate may include bases at the 3′ end to protect it against exodigestion. Examples of nucleotides that can be used to protect theacceptor template include, but are not limited to, ribonucleotides,thiophosphates, and nucleotide bases with or without modification.Alternatively, the reaction shown in FIG. 25 can be executed in a singlestep if, for example, a proper ratio of primer to exonuclease is used.As shown in FIG. 25, if the R2 reverse transcriptase dissociates fromthe DNA/RNA heteroduplex before completion of the jumping to theacceptor template, the product is overextended (3′ overhang). The 3′ to5′ exonuclease activity can then regenerate the bland end structure ofthe DNA/RNA duplex. In general, jumping to the acceptor template can becompleted by a multi-turnover mechanism, thus increasing the yield ofthe reaction;

FIG. 26 illustrates a BioAnalyzer trace data of Next-GenerationSequencing (NGS) libraries. Line 1 represents a fragmented RNA seqlibrary from plasma and line 2 represents a no-fragmented RNA seqlibrary from plasma (see, Example 15);

FIG. 27 and FIG. 28 illustrate methods of generating cell freedeoxyribonucleic acid (cfDNA) library;

FIG. 29 illustrates library preparation: pulling ribosomal RNA and/ortransfer RNA and/or PCR products using complementary oligonucleotideattached to magnetic beads or solid support to maximize sequencingcapacity;

FIG. 30 illustrates library preparation: oligonucleotide-guideddegradation of ribosomal RNA and/or transfer RNA and/or PCR products tomaximize sequencing capacity.

FIG. 31 illustrates a technology of the present disclosure that iscapable of capturing/targeting all species of RNA simultaneously. FIG.31 shows cell free RNA library that was obtained according to themethods of the present disclosure. The graph corresponds to Illuminasequencing results of library prepared from 20 ng cell free RNA (cfRNA).28357171 reads were analyzed, 91.9% mapped. Examples of captureditems/analytes included, but was not limited to, vault RNA, tRNA,srpRNA, sRNA, snRNA, snoRNA, scRNA, scaRNA, rRNA, RNA, long non-codingRNAs (lncRNAs), micro RNA (miRNA), macro lnc RNA, miscellaneous RNA, 3prime overlapping ncRNA, DNA, bidirectional promoter incRNA, lincRNA,MT_tRNA, MT_rNA, ribozyme, LTR, retroposon, and SINE.

DETAILED DESCRIPTION

While various embodiments of the disclosure have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions may occur to those skilled in theart without departing from the disclosure. It should be understood thatvarious alternatives to the embodiments of the disclosure describedherein may be employed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. In case of conflict,the present application including the definitions will control. Also,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Allpublications, patents and other references mentioned herein areincorporated by reference in their entireties for all purposes as ifeach individual publication or patent application were specifically andindividually indicated to be incorporated by reference, unless onlyspecific sections of patents or patent publications are indicated to beincorporated by reference. In order to further define the presentdisclosure, the following terms, abbreviations and definitions areprovided.

As used herein, the term “about” refers to variations in the numericalquantity that may occur, for example, through typical measuring andliquid handling procedures used for making concentrates or solutions inthe real world; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or to carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities. In some embodiments,the term “about” means within 10% of the reported numerical value, orwithin 5% of the reported numerical value, or within 20% of the reportednumerical value.

The indefinite articles “a” and “an” preceding an element or componentof the present disclosure are intended to be nonrestrictive regardingthe number of instances, i.e., occurrences of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The terms “anneal”, “hybridize” or “bind,” can be used interchangeablyherein to refer to the combining of one or more single-strandedpolynucleotide sequences, segments or strands, and allowing them to forma double-stranded molecule through base pairing. Two complementarysequences (e.g., ribonucleic acid (RNA) and/or deoxyribonucleic acid(DNA)) can anneal or hybridize by forming hydrogen bonds withcomplementary bases to produce a double-stranded polynucleotide or adouble-stranded region of a polynucleotide.

The term “subject” can be any animal which may benefit from the methodsof the disclosure, including, e.g., humans and non-human mammals, suchas primates, rodents, horses, dogs and cats. Subjects include withoutlimitation a eukaryotic organism, a mammal such as a primate, e.g.,chimpanzee or human, cow; dog; cat; a rodent, e.g., guinea pig, rat,mouse; rabbit; or a bird; reptile; or fish. Subjects specificallyintended for treatment using the methods described herein includehumans. A subject may be an individual or a patient.

As used herein, the term “primer extension reaction” generally refers tothe denaturing of a double-stranded nucleic acid, binding of a primer toone or both strands of the denatured nucleic acid, followed byelongation of the primer(s).

As used herein, the term “reaction mixture” generally refers to acomposition comprising reagents necessary to complete nucleic acidamplification (e.g., DNA amplification, RNA amplification), withnon-limiting examples of such reagents that include primer sets havingspecificity for target RNA or target DNA, DNA produced from reversetranscription of RNA, a DNA polymerase, a reverse transcriptase (e.g.,for reverse transcription of RNA), suitable buffers (includingzwitterionic buffers), co-factors (e.g., divalent and monovalentcations), dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG)),etc). In some cases, reaction mixtures can also comprise one or morereporter agents.

As used herein, a “reporter agent” generally refers to a compositionthat yields a detectable signal, the presence or absence of which can beused to detect the presence of amplified product.

As used herein, the term “target nucleic acid” generally refers to anucleic acid molecule in a starting population of nucleic acid moleculeshaving a nucleotide sequence whose presence, amount, and/or sequence, orchanges in one or more of these, are desired to be determined. A targetnucleic acid may be any type of nucleic acid, including DNA, RNA, andanalogues thereof.

The term “primer”, as used herein, refers to an oligonucleotide,occurring naturally as in a purified restriction digest or producedsynthetically that is characterized by an ability to be extended againsta template oligonucleotide, so that an oligonucleotide whose sequence iscomplementary to that of at least a portion of the template molecule islinked to the primer, when all are placed in the presence of nucleotidesat a suitable temperature and pH. However, the mere ability to be usedin this fashion does not require that primers be fully extended againsta template, and in some embodiments, primers are used only as a site forthe addition of a small number of non-templated nucleotides. Primerssuch as primer hexamers having a length of at least 6 nucleotides longcan be used. In some embodiments, a primer may be fluorescently labeled(e.g., 5′-/56FAM/TGATGACGAGGCATTTGGC/3′). In some embodiments, primershave a length within the range of about 6 to about 100 nucleotides, orin some embodiments from about 10 to about 70 nucleotides. In someembodiments, larger primers can be used. In some embodiments, randomprimers may be used. In some embodiments, a primer may be a randomprimer. In some embodiments, one or more primer(s) may be one or morerandom primer(s).

The term “one or more primer(s)” can comprise any number of primers orrandom primers. For example, “one or more primer(s)” can include atleast, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 primers orrandom primers. One or more primer(s) can include about 1 to about 2,about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 toabout 6, about 1 to about 7, about 1 to about 8, about 1 to about 9,about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1to about 25, about 1 to about 30, about 1 to about 35, about 5 to about15, about 3 to about 10, about 5 to about 20, about 10 to about 50,about 30 to about 100, or more than about 100 primers. One or moreprimer(s) can comprise any number of primers. For example, one or moreprimer(s) can include at least, at most, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000,14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000, 35000,40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000,90000, 95000, 100000, 150000, 200000, 250000, 300000, 350000, 400000,450000, 500000, 550000, 600000, 650000, 700000, 750000, 800000, 850000,900000, 950000, 1000000, 1500000, 2000000, 2500000, 3000000, 3500000,4000000, 4500000, 5000000, 5500000, 6000000, 6500000, 7000000, 7500000,8000000, 8500000, 9000000, 9500000, or 10000000 primers. One or moreprimer(s) can include about 10 to about 100, about 100 to about 1000,about 1000 to about 10,000, about 10,000 to about 100,000, about 100,000to about 1,000,000, or about 1,000,000 to about 10,000,000 primers.

The term “random primer,” as used herein, refers to a primer containinga random base sequence therein, and is intended to encompass primerswhether they consist partially or wholly of random base sequences.

In some embodiments, a primer may comprise an adaptor sequence. In someembodiments, the 5′ tail sequence of a primer comprises a sequence whichdoes not hybridize to a target (the adaptor sequence). The adaptorsequence may be selected such that it is the same in a variety ofprimers which have different 3′ target binding sequences (i.e., a“universal” 5′ tail sequence). This allows a single reporter probesequence to be used for detection of any desired target sequence, whichis an advantage in that synthesis of the reporter probe is more complexdue to the labeling. In some embodiments, a primer may comprise an RNAprimer. In some embodiments, a primer may comprise a DNA primer. In someembodiments, a primer may comprise an R2 RNA primer. In someembodiments, a primer may comprise one or more random primer(s).

As used herein, the tem “acceptor template” is synonymous to “acceptornucleic acid molecule.” In some embodiments, an acceptor nucleic acidmay be modified. In some embodiments, an acceptor nucleic acid moleculemay be modified at the 3′ end, for example to protect it from beingmistaken as an RNA primer. In some embodiments, the modification of theacceptor nucleic acid molecule may comprise a dideoxy 3′ end. In someembodiments, the modification may comprise a phosphorylated 3′ end. Insome embodiments, the phosphorylated 3′ end of a polynucleotide or of anacceptor nucleic acid molecule, which typically has a hydroxyl group onits 3′ end, can act as a 3′ block because extension by an enzyme of thepresent disclosure, or of DNA polymerase for example may be inhibited orligation by a ligase may be inhibited. Another non-limiting example of a3′ block includes the addition of a 3′ C3 spacer (three-carbon spacer)to the 3′ end of a polynucleotide which can function as an effectiveblocking agent against polymerase extension. Zhou, et al., Clin. Chem.,50: 1328-1335 (2004). Thus, the 3′ end can be blocked by the additionof, for example, a C3 spacer, a phosphate, an amine group (NH2), or anyother chemical modification that inhibits formation of a subsequentphosphodiester bond between the 3′ end of the polynucleotide and anothernucleotide.

An “overhang sequence,” as used herein, refers to a single strandedregion of nucleic acid extending from a double stranded region.

An “isolated” polynucleotide, as used herein, means a polynucleotidethat has been either removed from its natural environment, producedusing recombinant techniques, or chemically or enzymaticallysynthesized. A polynucleotide can also be purified, i.e., essentiallyfree from any other polynucleotides and associated cellular products orother impurities.

The term “polymerase” as used herein can refer to an enzyme that linksindividual nucleotides together into a strand, using another strand as atemplate. In some embodiments, the polymerase is a polymerase withediting capabilities. In some embodiments, the polymerase with editingcapabilities may be 3′ to 5′ exonuclease, T4 DNA polymerase, exonucleaseI, Phi29, Pfu, Vent, KOD, exonuclease III, and exonuclease T. Examplesof polymerases can include a DNA polymerase, an RNA polymerase, anRNA-directed DNA polymerase, reverse transcriptase, a polypeptide havingreverse transcriptase activity, or any variant thereof, a thermostablepolymerase, a wild-type polymerase, a modified polymerase, E. coli DNApolymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase PHI 29(phi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase,Pfu polymerase VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase,LA-Taq polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tnepolymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfipolymerase, Platinum Taq polymerases, Tbr polymerase, Tfl polymerase,Tth polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment,polymerase with 3′ to 5′ exonuclease activity, and variants, modifiedproducts and derivatives thereof. In some embodiments, the polymerasemay be a reverse transcriptase or a modified reverse transcriptase ofthe present disclosure. In some embodiments, the polymerase is a singlesubunit polymerase. The polymerase can have high processivity, namelythe capability of the polymerase to consecutively incorporatenucleotides into a nucleic acid template without releasing the nucleicacid template.

The term “reverse transcriptase” or RT refers to an enzyme with both anRNA-directed DNA polymerase and a DNA-directed DNA polymerase. RT refersto a group of enzymes having reverse transcriptase activity (e.g., thatcatalyze synthesis of DNA from an RNA template). In general, suchenzymes include, but are not limited to, retroviral reversetranscriptase, retrotransposon reverse transcriptase, retroplasmidreverse transcriptases, retron reverse transcriptases, bacterial reversetranscriptases, group II intron-derived reverse transcriptase, andmutants, variants or derivatives thereof. Non-retroviral reversetranscriptases include non-long terminal repeat (LTR) retrotransposonreverse transcriptases, retroplasmid reverse transcriptases, retronreverse transcriptases, and group II intron reverse transcriptases.Further bacterial reverse transcriptases are described by Simon D &Zimmerly S (2008) “A diversity of uncharacterized retroelements inbacteria” Nucleic Acids Res 36(22):7219-7229, and Kojima, K K &Kanehisa, M (2008) “Systematic survey for novel types of prokaryoticretroelements based on gene neighborhood and protein architecture” MolBiol Evol 25:1395-1404, which describe many classes of non-retroviralreverse transcriptases (i.e., retrons, group II introns, anddiversity-generating retroelements among others). Reverse transcriptasehas been used primarily to transcribe RNA into cDNA, which can then becloned into a vector for further manipulation or used in variousamplification methods such as polymerase chain reaction, nucleic acidsequence-based amplification (NASBA), transcription mediatedamplification (TMA), self-sustained sequence replication (3SR), diverseprimer extension reactions, 5′RACE, detection of chemical modificationsor other techniques that require synthesis of DNA using an RNA template.

Retroviral Reverse Transcriptase Enzymes

Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase contains asingle subunit of 78 kDa with RNA-dependent DNA polymerase and RNase Hactivity. This enzyme has been cloned and expressed in a fully activeform in E. coli (reviewed in Prasad, V. R., Reverse Transcriptase, ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 135(1993)).

Human Immunodeficiency Virus (HIV) reverse transcriptase is aheterodimer of p66 and p51 subunits in which the smaller subunit isderived from the larger subunit by proteolytic cleavage. The p66 subunithas both a RNA-dependent DNA polymerase and an RNase H domain, while thep51 subunit has only a DNA polymerase domain. Active HIV p66/p51 reversetranscriptase has also been cloned and expressed successfully in anumber of expression hosts, including E coli (reviewed in Le Grice, S.F. J., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory press, p. 163 (1993)). Within the HIV p66/p51heterodimer, the 51-kD subunit is catalytically inactive, and the 66-kDsubunit has both DNA polymerase and RNase H activity (Le Grice, S. F.J., et al., EMBO Journal 10:3905 (1991); Hostomsky, Z., et al., J.Virol. 66:3179 (1992)).

Members of the Avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptasefamily are also a heterodimers of two subunits, alpha (approximately 62kDa) and beta (approximately 94 kDa), in which the alpha subunit isderived from the beta subunit by proteolytic cleavage (reviewed inPrasad, V. R., Reverse Transcriptase, Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press (1993), p. 135). Members of this familyinclude, but are not limited to, Rous Sarcoma Virus (RSV) reversetranscriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase,Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reversetranscriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAVreverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) HelperVirus REV-A reverse transcriptase, Avian Sarcoma Virus UR2Helper VirusUR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAVreverse transcriptase, Rous Associated Virus (RAV) reversetranscriptase, and Myeloblastosis Associated Virus (MAV) reversetranscriptase, among others.

ASLV reverse transcriptase can exist in two additional catalyticallyactive structural forms, Ad and a (Hizi, A. and Joklik, W. K., J. Biol.Chem. 252: 2281 (1977)).

Sedimentation analysis suggests the presence of alpha/beta and beta/betaare dimers and that the a form exists in an equilibrium betweenmonomeric and dimeric forms (Grandgenett, D. P., et al., Proc. Nat.Acad. Sci. USA 70:230 (1973); Hizi, A. and Joklik, W. K., J. Biol. Chem.252:2281 (1977); and Soltis, D. A. and Skalka, A. M., Proc. Nat. Acad.Sci. USA 85:3372 (1988)). The ASLV alpha/beta and beta/beta reversetranscriptases are the only known examples of retroviral reversetranscriptase that include three different activities in the sameprotein complex: DNA polymerase, RNase H, and DNA endonuclease(integrase) activities (reviewed in Skalka, A. M., ReverseTranscriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress (1993), p. 193). The a form lacks the integrase domain andactivity.

Various forms of the individual subunits of ASLV reverse transcriptasehave been cloned and expressed. These include a 98-kDa precursorpolypeptide that is normally processed proteolytically to beta and a 4kDa polypeptide removed from the beta carboxy end (Alexander, F., etal., J. Virol. 61:534 (1987) and Anderson, D. et al., Focus 17:53(1995)), and the mature beta subunit (Weis, J. H. and Salstrom, J. S.,U.S. Pat. No. 4,663,290 (1987); and Soltis, D. A. and Skalka, A. M.,Proc. Nat. Acad. Sci. USA 85:3372 (1988)). (See also Werner S, and WohrlB. M., Eur. J. Biochem. 267:4740-4744 (2000); Werner S, and Wohrl B. M.,J. Virol. 74:3245-3252 (2000); Werner S, and Wohrl B. M., J. Biol. Chem.274:26329-26336 (1999).) Heterodimeric RSV alpha/beta reversetranscriptase has also been purified from E. coli cells expressing acloned RSV beta gene (Chemov, A. P., et al., Biomed. Sci. 2:49 (1991)).

Reverse Transcriptases of Non-Retroviral Origin

Reverse transcriptase enzymes may also be isolated from a large numberof mobile genetic elements which are not of retroviral origin. Suchmobile genetic elements are resident in the genomes of higher orderspecies and play a function role in life cycle of these mobile geneticelements. Mobile genetic elements are known to encode genes for reversetranscriptase enzymes (reviewed in Howard M Temin, Reverse Transcriptionin the Eukaryotic Genome: Retroviruses. Pararetroviruses,Retrotransposons, and Retrotranscripts, Mol. Biol. Evol. 2(6):455-468).These elements include, but are not limited, to retrotransposons.Retrotransposons include the non-long terminal repeat (LTR)retrotransposon and LTR mobile elements (e.g., TY3, TY5, non-LTR,LINE-L1, R2, R1). (Reviewed by Cordaux and Batzer, Nature Reviews,October 2009, volume 10, pp 691-703.).

As used herein, “non-LTR retrotransposon” refers to naturally occurringproteins encoded by non-LTR retrotransposons and polypeptide fragmentsthereof which possess reverse transcriptase activity, as well asproteins or polypeptides derived therefrom which contain one or moreamino acid substitutions that either enhance the reverse transcriptaseactivity thereof or have no deleterious effect thereon. A preferredclass of non-LTR retrotransposon are R2 proteins or polypeptides. Thus,as used herein, “R2 protein or R2 enzyme or polypeptide or a functionalfragment thereof” refers to naturally occurring proteins encoded by R2elements and polypeptide fragments thereof which possess reversetranscriptase activity, as well as proteins or polypeptides derivedtherefrom which contain one or more amino acid substitutions that eitherenhance the reverse transcriptase activity thereof or have nodeleterious effect thereon.

Retroelements, genetic elements that encode RTs, are divided into twomajor families denoted LTR-containing retroelements andnon-LTR-containing retroelements (Xiong Y, Eickbush T H (1990) “Originand evolution of retroelements based upon their reverse transcriptasesequences” EMBO J 9:3353-62). Non-LTR-retroelements are a diverse familyof RT-encoding elements that includes retroplasmids,non-LTR-retrotransposons, retrons, and mobile group II introns.

As used herein, the term polymerase “active fraction” is defined as afraction of enzyme with polymerase activity. For example, a reversetranscriptase active fraction (RT active fraction) is a fraction ofenzyme that has a reverse transcriptase activity.

As used herein, the terms “variant,” “modified,” “non-naturallyoccurring,” and “mutant” are synonymous and refer to a polypeptide orenzyme differing from a specifically recited polypeptide or enzyme byone or more amino acid insertions, deletions, mutations, andsubstitutions, created using, e.g., recombinant DNA techniques, such asmutagenesis. Guidance in determining which amino acid residues may bereplaced, added, or deleted without abolishing activities of interest,may be found by comparing the sequence of the particular polypeptidewith that of homologous polypeptides, e.g., yeast or bacterial, andminimizing the number of amino acid sequence changes made in regions ofhigh homology (conserved regions) or by replacing amino acids withconsensus sequences. In some embodiments, the terms “derivative,”“variant,” “modified,” “non-naturally occurring,” and “mutant” are usedinterchangeably.

The mutants of the present disclosure may be generated in accordancewith any suitable method, including, but not limited to, methodsdescribed and exemplified herein. Mutations, such as substitutions,insertions, deletions, and/or side chain modifications, may beintroduced into the nucleotide and amino acid sequences of the gene ofinterest using any suitable technique, including site-directedmutagenesis (Wu, ed., Meth. Enzymol. 217, Academic Press (1993)). Thelambda red recombinase method may be used to “knock out” genes (Datsenkoet al., PNAS USA 97: 6640-6645 (2000)). Permanent, marker-free, multiplegene disruptions may be created. Non-naturally occurring nucleotides andamino acids also may be used.

As used herein, “homologue” refers to a protein that is functionallyequivalent i.e. has the same enzymatic activity as an enzyme having anamino acid sequence of the specified sequence identification number, butmay have a limited number of amino acid substitutions, deletions,insertions or additions in the amino acid sequence. In order to maintainthe function of the protein, the substitutions may be conservativesubstitutions, replacing an amino acid with one having similarproperties.

In some embodiments, a homologue refers to a protein which has anidentity of at least about 25%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95% or at least about 99% with the amino acidsequence of SEQ ID NO corresponding to the protein. Algorithms fordetermining sequence identity include e.g. BLAST available through theNational Center for Biotechnology Information (NCBI). Sequences may bedetermined to be similar to a degree that indicates homology and thussimilar or identical function.

A polynucleotide encoding a homologue of each enzyme may be obtained byappropriately introducing substitution, deletion, insertion, and/oraddition to the DNA of the enzyme which is composed of a nucleotidesequence disclosed herein, using methods such as random mutagenesis andsite-specific mutagenesis (Nucleic Acid Res. 10, pp. 6487 (1982),Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2nd Edt.,Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRLPress pp. 200 (1991)). The polynucleotide encoding a homologue of eachenzyme may be introduced and expressed in a host to obtain thehomologue.

The term “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of thepresent disclosure may use either or both a heterologous or homologousencoding nucleic acid.

In some embodiments, a host cell may be selected from, and the modifiedor non-naturally occurring enzyme generated in, for example, bacteria,yeast, fungus or any of a variety of other organisms may be used as ahost organism.

In some embodiments, the host is not particularly restricted and theenzymatic activity or activities may be incorporated into any suitablehost organism using methods, for example, as described herein. In someembodiments, the host is selected from bacteria, yeast, algae,cyanobacteria, fungi, or a plant cell, or any combination thereof E.coli and S. cerevisiae are particularly useful host organisms since theyare well characterized microorganisms suitable for genetic engineering.

As used herein, “enzyme” includes proteins produced by a cell capable ofcatalyzing biochemical reactions. Further, unless context dictatesotherwise, as used herein “enzyme” includes protein fragments thatretain the relevant catalytic activity, and may include artificialenzymes synthesized to retain the relevant catalytic activity.

Each of the enzymes described herein may be attached to an additionalamino acid sequence as long as it retains an activity functionallyequivalent to that of the enzyme. As mentioned above, it is understoodthat each enzyme or a homologue thereof may be a (poly)peptide fragmentas long as it retains an activity functionally equivalent to that of theenzyme.

In some embodiments, the enzymes for use in compositions, methods andkits of the present disclosure include any enzyme having reversetranscriptase activity. Such enzymes include, but are not limited to,non-retroviral reverse transcriptases, retroviral reversetranscriptases, retrotransposon reverse transcriptases, non-LTRretrotransposons, R2 reverse transcriptases, LTR-retrotransposons,hepatitis B reverse transcriptases, cauliflower mosaic virus reversetranscriptases, bacterial reverse transcriptases, Tth DNA polymerases,Taq DNA polymerases (Saiki, R. K., et al., Science 239:487-491 (1988);U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne DNA polymerases (PCTPublication No. WO 96/10640), Tma DNA polymerases (U.S. Pat. No.5,374,553) and mutants, fragments, variants or derivatives thereof. Insome embodiments, reverse transcriptases for use in the presentdisclosure include retroviral reverse transcriptases such as M-MLVreverse transcriptase, AMV reverse transcriptase, RSV reversetranscriptase, RAV reverse transcriptase, MAV reverse transcriptase, andgenerally ASLV reverse transcriptases. Mutant reverse transcriptasescan, for example, be obtained by mutating the gene or genes encoding thereverse transcriptase of interest by site-directed or randommutagenesis. Such mutations may include point mutations, deletionmutations, insertional mutations, and truncations. For example, one ormore point mutations (e.g., substitution of one or more amino acids withone or more different amino acids) may be used to construct mutantreverse transcriptases for use in the present disclosure.

In some embodiments, the enzyme is selected and/or engineered to exhibithigh fidelity with low error rates. The fidelity of a nucleotidepolymerase is typically measured as the error rate, i.e., the frequencyof incorporation of a nucleotide in a manner that violates the widelyknown Watson-Crick base pairing rules. The fidelity or error rate of apolymerase (e.g., DNA polymerase) may be measured using any suitableassay. See, for example, Lundburg et al., 1991 Gene, 108:1-6. The term“fidelity” can be used to refer to the accuracy of polymerization, orthe ability of the polymerase to discriminate correct from incorrectsubstrates, (e. g., nucleotides) when synthesizing nucleic acidmolecules (e. g. RNA or DNA) which are complementary to a template. Thehigher the fidelity of an enzyme, the less the enzyme misincorporatesnucleotides in the growing strand during nucleic acid synthesis; thatis, an increase or enhancement in fidelity results in a more faithfulpolymerase having decreased error rate (decreased misincorporationrate). In some embodiments, the misincorporation error rate is at mostabout 10-2, 10-4, 10-6, or 10-8.

In some embodiments, the non-naturally occurring or modified enzyme(e.g., non-naturally occurring or modified reverse transcriptase,non-naturally occurring or modified non-LTR retrotransposon,non-naturally occurring or modified R2 reverse transcriptase) or amodified polypeptide having reverse transcriptase activity exhibits amisincorporation error rate of equal to or less than about 50%, equal toor less than about 45%, equal to or less than about 40%, equal to orless than about 35%, equal to or less than about 30%, equal to or lessthan about 25%, equal to or less than about 20%, equal to or less thanabout 15%, equal to or less than about 10%, equal to or less than about9%, equal to or less than about 8%, equal to or less than about 7%,equal to or less than about 6% equal to or less than about 5%, equal toor less than about 4%, equal to or less than about 3%, equal to or lessthan about 2%, equal to or less than about 1%, equal to or less thanabout 0.01%, equal to or less than about 0.001%, equal to or less thanabout 0.0001%, equal to or less than about 0.00001%, equal to or lessthan about 0.000001%, or equal to or less than about 0.0000001%.

In some embodiments, the non-naturally occurring or modified enzyme(e.g., non-naturally occurring or modified reverse transcriptase,non-naturally occurring or modified non-LTR retrotransposon,non-naturally occurring or modified R2 reverse transcriptase) or amodified polypeptide having reverse transcriptase activity generates oneor more nucleic acid (e.g., cDNA) molecule(s) complementary to atemplate at an error rate that is at least about 10000 times lower, atleast about 1500 times lower, at least about 1000 times lower, at leastabout 500 times lower, at least about 100 times lower, at least about 95times lower, at least about 90 times lower, at least about 85 timeslower, at least about 80 times lower, at least about 75 times lower, atleast about 70 times lower, at least about 65 times lower, at leastabout 60 times lower, at least about 55 times lower, at least about 50times lower, at least about 45 times lower, at least about 40 timeslower, at least about 35 times lower, at least about 30 times lower, atleast about 25 times lower, at least about 20 times lower, at leastabout 15 times lower, at least about 10 times lower, at least about 9times lower, at least about 8 times lower, at least about 7 times lower,at least about 6 times lower, at least about 5 times lower, at leastabout 4 times lower, at least about 3 times lower, at least about 2times lower, or at least about 1 time lower than the unmodified ornaturally occurring enzyme or unmodified polypeptide having reversetranscriptase activity.

In some embodiments, the sequencing error rate will be equal to or lessthan about 1 in 100,000 bases. In some embodiments, the error rate ofnucleotide sequence determination is equal to or less than about 1 in 10bases, 1 in 20 bases, 3 in 100 bases, 1 in 100 bases, 1 in 1000 bases,and 1 in 10,000 bases.

The terms “polynucleotides”, “nucleic acid”, “nucleotides” and“oligonucleotides” can be used interchangeably. They can refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, fragments, or analogs thereof. The following arenon-limiting examples of polynucleotides: coding or non-coding regionsof a gene or gene fragment, loci (locus) defined from linkage analysis,exons, introns, messenger RNA (mRNA), transfer RNA, transfer-messengerRNA, ribosomal RNA, antisense RNA, small nuclear RNA (snRNA), smallnucleolar RNA (snoRNA), micro-RNA (miRNA), small interfering RNA(siRNA), ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.A nucleic acid described herein can contain phosphodiester bonds. Insome embodiments, the nucleic acids can be DNA (including, e.g., genomicDNA, mitochondrial DNA, and cDNA), RNA (including, e.g., mRNA and rRNA)or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A polynucleotide isintended to encompass a singular nucleic acid as well as plural nucleicacids The polynucleotide may be composed of any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, polynucleotides may be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions.

Ribosomal RNAs can make up as much as 80% or more of the total RNA in asample. It is often desirable to separate mRNA from rRNA because rRNAcan adversely affect the quantitative analysis of mRNA. One approach toseparating rRNA from mRNA is to deplete the rRNA from the sample. Oneexample, is the hybridization of rRNA molecules using oligonucleotides,for example, oligonucleotides homologous to the 17S rRNA, 18S rRNA, or28S rRNA in the case of eukaryotic rRNAs, or to the 16S rRNA or 23S rRNAin the case of bacterial rRNA. The oligonucleotides are designed suchthat they can be “captured” and the hybridization product removed fromthe sample. For example, the oligonucleotides may be immobilized on asurface such as a column or a bead. MICROBExpress (Registered Trademark)and MICROBEnrich (Registered Trademark) (Ambion, Austin, Tex.) areexamples of commercially available kits for the depletion of rRNA.Methods and compositions for the depletion or rRNA from a sample aredescribed in U.S. application Ser. No. 10/029,397, which is incorporatedby reference. The poly(A) tail at the 3′ end of most eukaryotic mRNAscan be used to separate these molecules away from rRNA and othernon-mRNA species that lack this poly(A) tail. In some embodiments, themethod of the present disclosure comprises depleting ribosomal RNA, suchas by hybridization of an oligonucleotide to an rRNA. In someembodiments, the method of the present disclosure comprises depletingrRNA and/or tRNA by oligonucleotide probe-guided endonucleoliticcleavage of at least one rRNA and/or tRNA sequence. In some embodiments,depletion can be partial or a complete depletion. In some embodiments,depletion comprises decreasing the number of rRNA and/or tRNA from asample. In some embodiments, the present disclosure relates to methodsof depleting rRNA and/or transfer RNA (tRNA). In some embodiments, thepresent disclosure relates to depleting at least one transfer RNA (tRNA)from one or more template nucleic acid molecules. In some embodiments,depleting rRNA and/or tRNA occurs prior to annealing of a primer (e.g.,one or more primers) to a template.

In one embodiment, the present disclosure relates to a method ofdepleting ribosomal and/or transfer RNA from a sample for librarysequencing. In some embodiments, the method comprises providing a samplecomprising RNA. In some embodiments, the RNA comprises ribosomal RNA(rRNA) and/or transfer RNA (tRNA). In some embodiments, the methodcomprises performing a polymerase chain reaction (PCR) to convert therRNA and/or tRNA to double stranded DNA (dsDNA). In some embodiments,the method comprises partial or full (complete) amplification. In someembodiments, the method further comprises introducing a complexcomprising a nuclease and/or a polynucleotide encoding a nuclease and atleast one specifically designed guide oligonucleotide. In someembodiments, the at least one guide oligonucleotide comprises a sequencecomplementary to at least one rRNA and/or at least one tRNA. In someembodiments, the at least one guide oligonucleotide comprises at leastone sequence complementary to at least one dsDNA. In some embodiments,the nuclease or polynucleotide encoding the nuclease cleaves at leastone strand of the dsDNA. In some embodiments, the nuclease orpolynucleotide encoding the nuclease cleaves the rRNA and/or the tRNAand/or the dsDNA, thereby depleting the rRNA and/or the tRNA from thesample. In some embodiments, the nuclease is Cas9 or the polynucleotideencodes Cas9 or a functional variant thereof. In some embodiments, themethod comprises denaturing the dsDNA into single-stranded DNA (ssDNA)strands. In some embodiments, the method further comprises introducingat least one oligonucleotide (e.g., specifically designedoligonucleotide) comprising a binding molecule and at least one sequencecomplementary to at least one ssDNA strand to form a hybridized complexof the oligonucleotide and the at least one ssDNA strand. In someembodiments, the method comprises immobilizing the hybridized complex toat least one solid support. In some embodiments, immobilizing thehybridized complex causes the hybridized complex to be removed from asample. In some embodiments, the solid support comprises streptavidin.In some embodiments, the method comprises introducing at least oneoligonucleotide (e.g., specifically designed oligonucleotide) comprisinga binding molecule and at least one sequence complementary to at leastone rRNA and/or tRNA to form a complex comprising the oligonucleotideand the at least one rRNA and/or tRNA. In some embodiments, the methodfurther comprises immobilizing the complex to at least one solidsupport. In some embodiments, immobilizing the complex causes thecomplex to be removed from a sample. In some embodiments, the solidsupport comprises streptavidin. In some embodiments, the bindingmolecule is biotin.

In some embodiments, any of the method of the present disclosurecomprises a nuclease or polynucleotide encoding the nuclease. In someembodiments, the nuclease or polynucleotide encoding the nucleasecleaves at least one strand of a dsDNA. In some embodiments, cleavage ofdsDNA is an intermediate product of library preparation. In someembodiments, cleavage of dsDNA includes rRNA and/or tRNA (codingsequences). In some embodiments, oligo-guided nucleolitic cleavage maynot or does not require denaturing dsDNA. In some embodiments, notrequiring dsDNA denaturation is a significant improvement of the presentdisclosure. In some embodiments, the nuclease or a polynucleotideencoding the nuclease is Cas9, or a polynucleotide encoding Cas9, or afunctional variant thereof.

In one embodiment, the present disclosure relates to a method comprisingadding a nuclease complex to a plurality of double-stranded nucleic acidmolecules. In some embodiments, the nuclease complex comprises a Cas9nuclease or a functional variant thereof and at least one syntheticguide oligonucleotide. In some embodiments, the synthetic guideoligonucleotide is complementary to a ribosomal ribonucleic acid (rRNA)and/or transfer ribonucleic acid (tRNA) region. In some embodiments, theregion is in at least one double-stranded nucleic acid molecule. In someembodiments, the method comprises permitting the complex to cleave anrRNA and/or a tRNA region. In some embodiments the region is present inat least one double-stranded nucleic acid molecule. In some embodiments,the method provides at least one cleaved double-stranded nucleic acidmolecule. In some embodiments, the method comprises subjecting the atleast one cleaved double-stranded nucleic acid molecule or derivativethereof to sequencing (e.g., nucleic acid sequencing). In someembodiments, the method comprises sequencing a nucleic acid sequence ofat least one double-stranded nucleic acid molecule lacking an rRNAand/or a tRNA region. In some embodiments, the method comprisessequencing a nucleic acid sequence of at least one double-strandednucleic acid molecule comprising an rRNA and/or a tRNA region. In someembodiments, the method comprises sequencing a mixture of nucleic acidsequence wherein the mixture comprises at least one double-strandednucleic acid molecule comprising an rRNA and/or a tRNA region and atleast one double-stranded nucleic acid molecule lacking an rRNA and/or atRNA region.

In one embodiment, the present disclosure relates to a method ofproducing a cell free deoxyribonucleic acid (cfDNA) library comprising:providing a sample comprising cfDNA; denaturing the cfDNA to produce asingle stranded DNA (ssDNA) sample; introducing, in the presence ofnucleotides and/or a catalytic metal, a complex comprising a template, aprimer, and a reverse transcriptase to the ssDNA sample, wherein thereverse transcriptase extends the primer on the template andsubsequently template jumps to the ssDNA sample to produce a doublestranded DNA (dsDNA) sample, and wherein the dsDNA comprises at leastone nick between the template and the ssDNA; introducing a polymerasecomprising a 3′-to-5′ exonuclease activity to generate a dsDNA withblunt ends and/or a 3′-overhang; introducing an asymmetric adaptercomprising a nucleic acid duplex with a single-stranded overhang at the5′ end, wherein the asymmetric adapter is ligated to the 5′ end of thedsDNA, and wherein the single-stranded overhang comprises a sequencecomplementary to at least one polymerase chain reaction (per)amplification primer; and performing a per reaction to amplify only onestrand of the dsDNA.

In one embodiment, the present disclosure relates to a method ofproducing a cell free deoxyribonucleic acid (cfDNA) library. In someembodiments, the method comprises providing a sample comprising cfDNA.In some embodiments, the method comprises denaturing the cfDNA toproduce a single stranded DNA (ssDNA) sample. In some embodiments, themethod comprises introducing a terminal deoxynucleotidyl transferase(TdT) and a deoxyadenosine triphosphate (dATP) to the ssDNA sample togenerate a poly(A) and/or a poly(C) tail. In some embodiments, themethod comprises introducing a non-extendable nucleotide. In someembodiments, the method comprises annealing a complex comprising aprimer and a first adapter to the tail of the ssDNA sample. In someembodiments, the complex comprises a sequence complementary to the tail.In some embodiments, the method comprises introducing, in the presenceof nucleotides and/or a catalytic metal, a reverse transcriptase (e.g.,a modified reverse transcriptase) and a complex comprising an acceptorand a second adapter to produce a double strand DNA (dsDNA) sample. Insome embodiments, the nucleotides comprise degradable nucleotides. Insome embodiments, the reverse transcriptase extends the primer andsubsequently template jumps to the complex to continue extension. Insome embodiments, the complex comprises a nucleotide block to preventthe reverse transcriptase from reaching the end of the complex andjumping to another complex. In some embodiments, the dsDNA comprises anoriginal strand and a copy strand. In some embodiments, the originalstrand comprises at least one nick between the complex and the ssDNA. Insome embodiments, the copy strand comprises at least one degradablenucleotide. In some embodiments, the method further comprisesintroducing a polymerase comprising a 3′-to-5′ exonuclease activity togenerate a dsDNA with blunt ends or a 3′-overhang, and/or a DNA ligaseto ligate the at least one nick. In some embodiments, the method furthercomprises introducing at least one uracil-DNA glycosylase to degrade atleast one degradable nucleotide. In some embodiments, the method furthercomprises performing a polymerase chain reaction (PCR) comprising aprimer (e.g., at least a first primer and/or at least a first and asecond primer) to amplify the original strand. In some embodiments, theprimer (e.g., the first primer) comprises a sequence complementary tothe first adapter and the second primer comprises a sequencecomplementary to the second adapter.

In one embodiment, the present disclosure relates to a method ofproducing a library for sequencing. In some embodiments, the methodcomprises providing a sample comprising cell free ribonucleic acid(cfRNA). In some embodiments, the method comprises subjecting the sampleto high temperature. In some embodiments, the high temperature issufficient to allow for transphosphorylation of the RNA (e.g., cfRNA).In some embodiments, the method further comprises introducing aphosphatase. In some embodiments, the phosphatase can convert aphosphate moiety of an RNA to a 3′-hydroxyl group. In some embodiments,the method further comprises introducing an adenosine triphosphate and apolymerase to generate a poly(A) tail on the 3′-hydroxyl group of theRNA. In some embodiments, the method further comprises introducing, inthe presence of nucleotides, a primer, an acceptor, and a reversetranscriptase. In some embodiments, the primer comprises a sequencecomplementary to the poly(A) tail thereby annealing to the poly(A) tail.In some embodiments, the reverse transcriptase extends the primer andsubsequently template jumps to the acceptor to continue extension. Insome embodiments, the method further comprises introducing at least onesolid support to immobilize excess primer and non-specific primerproducts to the at least one solid support, thereby removing the excessprimer and the non-specific primer products from the sample. In someembodiments, the method further comprises performing a polymerase chainreaction (PCR) reaction to amplify the RNA. In some embodiments, themethod further comprises using an isothermal amplification reaction.

In one embodiment, the present disclosure relates to a method forpreparing a nucleic acid library for sequencing. In some embodiments,the method comprises obtaining a plurality of nucleic acid molecules. Insome embodiments, the method comprises inducing a non-enzymaticintramolecular transphosphorylation of at least one nucleic acidmolecule (e.g., in the plurality of nucleic acid molecules). In someembodiments, the non-enzymatic intramolecular transphosphorylation canoccur by increasing temperature (e.g., increase the temperature of aplurality of nucleic acid molecules). In some embodiments, non-enzymaticintramolecular transphosphorylation and/or an increase of temperatureresults in a nucleic acid molecule having a free 5′-phosphate moiety(e.g., a plurality of nucleic acid molecules can have a free5′-phosphate moiety). In some embodiments, the method comprises adding aphosphatase to the nucleic acid molecules with the free 5′-phosphatemoiety. In some embodiments, the phosphatase converts one or more of thefree 5′-phosphate moieties to a hydroxyl group. In some embodiments,this results in a plurality of nucleic acid molecules to have a freehydroxyl group. In some embodiments, the method comprises mixing (e.g.,in the presence of an amount of adenosine triphosphates) a plurality ofnucleic acid molecules and a polymerase. In some embodiments, thepolymerase generates a poly(A) tail on the free hydroxyl group. In someembodiments, the method comprises mixing, in the presence ofnucleotides, (i) one or more primers comprising a sequence complementaryto said poly(A) tail; (ii) one or more acceptor nucleic acid molecules;and (iii) a modified reverse transcriptase. In some embodiments, themodified reverse transcriptase generates a plurality of continuouscomplementary deoxyribonucleic acid molecule by reverse transcribing asequence of an annealed template nucleic acid molecule, migrating to anacceptor nucleic acid molecule, and reverse transcribing a sequence ofsaid acceptor nucleic acid molecule. In some embodiments, the methodcomprises adding at least one solid support. In some embodiments, thesolid support immobilizes an excess of the one or more primerscomprising a sequence complementary to said poly(A) tail. In someembodiments, the method comprises performing a polymerase chain reaction(PCR) reaction. In some embodiments, the method comprises performing anisothermal amplification.

In one embodiment, the present disclosure relates to a method ofproducing a library for sequencing. In some embodiments, the methodcomprises providing a sample comprising at least one nucleic acidmolecule (such as ribonucleic acid (e.g., cfRNA)). In some embodiments,the method comprises subjecting the sample (or the nucleic acidmolecule) to high temperature sufficient to allow fortransphosphorylation of the nucleic acid molecule (e.g., RNA). In someembodiments, the method further comprises adding a catalytic metal(e.g., magnesium) and/or a polyamine. In some embodiments, the methodcomprises introducing a phosphatase to convert a phosphate moiety of anucleic acid molecule (e.g., RNA) to a 3′-hydroxyl group. In someembodiments, the method comprises introducing an adenosine triphosphateand a polymerase to generate a poly(A) tail. In some embodiments, thepoly(A) tail is generated on the 3′-hydroxyl group of the nucleic acidmolecule (e.g., RNA). In some embodiments, the method comprisesintroducing, in the presence of nucleotides, a primer, an acceptor, anda reverse transcriptase to the sample or to the nucleic acid molecule.In some embodiments, the primer comprises a sequence complementary tothe poly(A) tail thereby annealing to the poly(A) tail. In someembodiments, the reverse transcriptase extends the primer andsubsequently template jumps to the acceptor to continue extension. Insome embodiments, the method comprises introducing at least one solidsupport. In some embodiments, the solid support immobilizes excessprimer and non-specific primer products. In some embodiments, the excessprimer and the non-specific primer products is removed from thesample/mixture. In some embodiments, the method further comprisesperforming a polymerase chain reaction (PCR) reaction to amplify thenucleic acid molecule (e.g., RNA). In some embodiments, the methodfurther comprises an isothermal amplification reaction.

In one embodiment, the present disclosure relates to a method ofdepleting rRNA and/or tRNA and/or to a method comprising reversetranscribing at least one nucleic acid molecule by performing anamplification reaction. In some embodiments, at least one nucleic acidmolecule is rRNA and/or tRNA. In some embodiments, the amplificationreaction provides a plurality of double-stranded nucleic acid molecules(e.g., cDNA). In some embodiments, the method comprises adding anuclease or a polypeptide comprising a nuclease and/or a guideoligonucleotide. In some embodiments, the method comprises adding acomplex comprising a nuclease or a polypeptide comprising a nuclease anda guide oligonucleotide. In some embodiments, the nuclease orpolypeptide comprising the nuclease is Cas9 or a polypeptide comprisingCas9. In some embodiments, the guide oligonucleotide is complementary toregion of a nucleic acid molecule. In some embodiments, the guideoligonucleotide is complementary to a pre-determined region in at leastone nucleic acid molecule (e.g., double-stranded nucleic acid molecule).In some embodiments, the oligonucleotide directs the nuclease (e.g.,Cas9) to the site of cleavage. In some embodiments, the complexcomprising a nuclease and a guide oligonucleotide cleaves a region or apre-determined region of the nucleic acid molecule. In some embodiments,the cleavage is at a gene. In some embodiments, the method comprisessequencing the cleaved nucleic acid molecules (e.g., cleaved doublestranded nucleic acid molecules) (e.g., sequencing a library of cleavednucleic acid molecules). In some embodiments the double-stranded nucleicacid molecules comprises sequences derived from an rRNA, a tRNA, or bothan rRNA and a tRNA. In some embodiments, the pre-determined region of atleast one double-stranded nucleic acid molecule is a region of an rRNA,a tRNA, or both (e.g., a combination). In some embodiments, thepre-determined region is a region of an rRNA. In some embodiments, thepre-determined region is a region of a tRNA. In some embodiments themethod does not require denaturation of nucleic acid molecules. In someembodiments, the method does not require denaturation of the pluralityof double-stranded nucleic acid molecules. In some embodiments, adouble-stranded nucleic acid molecule comprises a sequence derived froman rRNAs and/or a tRNA prior to sequencing. In some embodiments, thedouble-stranded nucleic acid molecules comprise a cDNA.

In one embodiment, the present disclosure relates to a method forpreparing a concatemer of nucleic acid molecules. In some embodiments,the method comprises processing ends of a plurality of double-strandednucleic acid molecules. In some embodiments, the method comprises addinga first plurality of adaptor molecules to the plurality of doublestranded nucleic acid molecules. In some embodiments, the firstplurality of adaptor molecules comprise one or more overhang sequences.In some embodiments, at least two of the one or more overhang sequencesare complementary to each other. In some embodiments, the methodprovides a first plurality of adaptor connected double-stranded nucleicacid molecules. In some embodiments, the method comprises adding apolymerizing enzyme (e.g., adding a polymerase enzyme to the firstplurality of adaptor connected double-stranded nucleic acid molecules).In some embodiments, adding a polymerase enzyme is in the absence of aprimer. In some embodiments, the method does not comprise adding aprimer. In some embodiments, the polymerizing enzyme forms a first setof adaptor connected double-stranded nucleic acid concatemers. In someembodiments, forming a first set of adaptor connected double-strandednucleic acid concatemers is by joining two or more adaptor connecteddouble-stranded nucleic acid molecules by the one or more overhangsequences. In some embodiments, the method comprises adding a secondplurality of adaptor molecules to the first set (e.g., first adaptormolecules). In some embodiments, the second plurality of adaptormolecules comprise one or more overhang sequences. In some embodiments,at least two of the one or more overhang sequences are complementary toeach other. In some embodiments, the method provides a second set ofadaptor connected double-stranded nucleic acid molecules. In someembodiments, any one of the previous embodiments can be repeated with aset of adaptor molecules to yield a concatemer comprising apredetermined average length.

In one embodiment, the present disclosure relates to a method forpreparing a concatemer of nucleic acid molecules. In some embodiments,the method comprises subjecting at least one nucleic acid moleculeand/or a plurality of double-stranded nucleic acid molecules toend-repair. In some embodiments, the method comprises adding at leastone or a plurality of adaptor molecules to the at least one nucleic acidmolecule and/or the plurality of double-stranded nucleic acid molecules.In some embodiments, adding at least one or a (first) plurality ofadaptor molecules to the at least one nucleic acid molecule and/or theplurality of double stranded nucleic acid molecules comprises ligation.In some embodiments, adding at least one or a (first) plurality ofadaptor molecules to the at least one nucleic acid molecule and/or theplurality of double stranded nucleic acid molecules comprises a reversetranscriptase (e.g., R2 reverse transcriptase, or a modified reversetranscriptase). In some embodiments, In some embodiments, the at leastone or a plurality of adaptor molecules comprise one or more overhangsequences. In some embodiments, at least two overhang sequences arecomplementary to each other (e.g., thereby providing a (first) pluralityof adaptor connected double-stranded nucleic acid molecules). In someembodiments, the at least one or a plurality of adaptor moleculescomprise a sequence (e.g., overhang sequence) that attaches/ligates tothe 3′ end of the nucleic acid molecule and/or a sequence (e.g.,overhang sequence) that attaches/ligates to the 5′ end of the nucleicacid molecule. In some embodiments, the nucleic acid molecule comprisesadaptors on both the 3′ and the 5′ end. In some embodiments, the adaptorthat binds to the 3′ end is complementary to the adaptor that binds tothe 5′ end. In some embodiments, the sequence of the adaptors isunknown. In some embodiments, the sequence of the adaptors ispre-determined. In some embodiments, the adaptor serves as a templateand/or as a primer. In some embodiments, the adaptor that binds to the3′ end of one nucleic acid molecule can bind to an adaptor on the 5′ endof another nucleic acid molecule. In some embodiments, the methodfurther comprises adding a polymerase enzyme to the adaptor connected toa nucleic acid molecule. In some embodiments, the method furthercomprises adding a polymerase to the (first) plurality of adaptorconnected double-stranded nucleic acid molecules. In some embodiments,the polymerase is added in the absence of a primer. In some embodiments,the polymerase enzyme forms a first set of adaptor connecteddouble-stranded nucleic acid concatemers by joining two or more adaptorconnected double-stranded nucleic acid molecules by the one or moreoverhang sequences. In some embodiments, the polymerase permits that theadaptor connected to the nucleic acid molecule form concatemers. In someembodiments, the method comprises adding a second plurality of adaptormolecules to the first set. In some embodiments, the second plurality ofadaptor molecules comprise one or more overhang sequences. In someembodiments, the at least two overhang sequences are complementary toeach other. In some embodiments, a second set of adaptor connecteddouble-stranded nucleic acid molecules is formed. In some embodiments,the concatemer length or the number of attached templates can bedetermined, for example, by tagging the adaptors with modifiednucleotides (e.g., by introducing methylated nucleotides or by insertingdUTP). In some embodiments the length of the concatemer can be regulatedbased on the ratio between modified/unmodified adaptors. In someembodiments the adaptor sequences can serve as a homology priminglocation (annealed to the homology spot ssDNA fragments serve astemplate and primer). In some embodiments, the method comprisesamplifying the concatemers by PCR or isothermal reaction. In someembodiments, the reaction in the PCR undergoes a selected number ofcycles (the more cycles, the longer the concatemer) or time (isothermalamplification). In some embodiments, the reaction is stopped and the(long) dsDNA concatemers are ligated with two unique dsDNA adaptors. Insome embodiments, the length of the concatemer can be manipulated. Insome embodiments, the length of the concatemer can be determined atleast based on the number of PCR cycles, and/or the amount of time(e.g., in an isothermal amplification), and/or based on the modifiednucleotide present in the adaptor. In some embodiments, the adaptorcomprises a unique molecular identifier sequence (UMI). In someembodiments, the polymerase enzyme joins two or more adaptor connecteddouble-stranded nucleic acid molecules in a PCR or isothermalamplification reaction. In some embodiments, the adaptor comprises atleast one modified nucleotide.

In some embodiments, nucleic acid from a biological sample obtained froma subject is amplified. In some cases, the biological sample is obtaineddirectly from the subject. In some embodiments, a biological sampleobtained directly from a subject refers to a biological sample that hasbeen further processed after being obtained from the subject. In someembodiments, a biological sample obtained directly from a subject refersto a biological sample that has not been further processed after beingobtained from the subject, with the exception of any approach used tocollect the biological sample from the subject for further processing.For example, blood is obtained directly from a subject by accessing thesubject's circulatory system, removing the blood from the subject (e.g.,via a needle), and entering the removed blood into a receptacle. Thereceptacle may comprise reagents (e.g., anti-coagulants) such that theblood sample is useful for further analysis. In another example, a swabmay be used to access epithelial cells on an oropharyngeal surface ofthe subject. After obtaining the biological sample from the subject, theswab containing the biological sample can be contacted with a fluid(e.g., a buffer) to collect the biological fluid from the swab.

In some embodiments, a biological sample has been purified. In someembodiments, a biological sample has not been purified. In someembodiments, the nucleic acid of a biological sample has not beenextracted when the biological sample is provided to a tube. For example,the RNA or DNA in a biological sample may not be extracted from thebiological sample when providing the biological sample to a tube. Insome embodiments, a target nucleic acid (e.g., a target RNA or targetDNA) present in a biological sample may not be concentrated prior toproviding the biological sample to a reaction vessel (e.g., a tube). Anysuitable biological sample that comprises nucleic acid may be obtainedfrom a subject.

The present disclosure relates to a non-naturally occurring or modifiedenzyme (e.g., a non-naturally occurring or modified reversetranscriptase, modified reverse transcriptase) or a modified polypeptidehaving reverse transcriptase activity that has an improved enzymeproperty compared to a naturally occurring or wild type or unmodifiedenzyme (e.g., a wild type reverse transcriptase) or unmodifiedpolypeptide having reverse transcriptase activity. In some embodiments,the non-naturally occurring or modified enzyme is an enzyme with reversetranscriptase activity. In some embodiments, the non-naturally occurringor modified enzyme is a modified reverse transcriptase. In someembodiments, the non naturally occurring or modified enzyme is amodified non-retroviral reverse transcriptase. In some embodiments, thenon-naturally occurring or modified enzyme is a modified non-LTRretrotransposon. In some embodiments, the non-naturally occurring ormodified enzyme is a modified R2 reverse transcriptase. In someembodiments, a non-naturally occurring or modified enzyme or a modifiedpolypeptide having reverse transcriptase activity can amplify a templatenucleic acid molecule at a processivity of at least about 80% per base,of at least about 85% per base, of at least about 88% per base, of atleast about 89% per base, of at least about 90% per base, of at leastabout 91% per base, of at least about 92% per base, of at least about93% per base, of at least about 94% per base, of at least about 95% perbase, of at least about 96% per base, of at least about 97% per base, ofat least about 98% per base, of at least about 99% per base, of at leastabout 99.5% per base, or of about 100% per base.

In some embodiments, a non-naturally occurring or modified enzyme or amodified polypeptide having reverse transcriptase activity can amplifyor is capable of amplifying a template nucleic acid molecule at aprocessivity measured at a temperature of between about 12° C. and about40° C. In some embodiments, the temperature is between about 10° C. andabout 35° C., between about 12° C. and about 30° C., between about 25°C. and about 40° C., or between about 12° C. and about 42° C. In someembodiments, the temperature is between about 8° C. to about 50° C.,between about 2° C. to about 60° C., between about 8° C. to about 42°C., between about 6° C. to about 32° C., or between about 7° C. to about35° C.

In some embodiments, a non-naturally occurring or modified enzyme or amodified polypeptide having reverse transcriptase activity can amplifyor is capable of amplifying a template nucleic acid molecule at aprocessivity of at least about 80% per base at a temperature at about orat most about 4° C., at about or at most about 8° C., at about or atmost about 12° C., at about or at most about 15° C., at about or at mostabout 20° C., at about or at most about 25° C., at about or at mostabout 30° C., at about or at most about 35° C., at about or at mostabout 40° C., or at about or at most about 42° C.; of at least about 85%per base at a temperature of about or at most about 12° C., at about orat most about 15° C., at about or at most about 20° C., at about or atmost about 25° C., at about or at most about 30° C., at about or at mostabout 35° C., at about or at most about 40° C., at about or at mostabout 42° C., at about or at most about 45° C., at about or at mostabout 50° C.; of at least about 89% per base at a temperature at aboutor at most about 4° C., at about or at most about 8° C., at about or atmost about 12° C., at about or at most about 15° C., at about or at mostabout 20° C., at about or at most about 25° C., at about or at mostabout 30° C., at about or at most about 35° C., at about or at mostabout 40° C., or at about or at most about 42° C.; of at least about 90%per base at a temperature of about or at most about 12° C., at about orat most about 15° C., at about or at most about 20° C., at about or atmost about 25° C., at about or at most about 30° C., at about or at mostabout 35° C., at about or at most about 40° C., at about or at mostabout 42° C., at about or at most about 45° C., at about or at mostabout 50° C.; of at least about 91% per base at a temperature at aboutor at most about 4° C., at about or at most about 8° C., at about or atmost about 12° C., at about or at most about 15° C., at about or at mostabout 20° C., at about or at most about 25° C., at about or at mostabout 30° C., at about or at most about 35° C., at about or at mostabout 40° C., or at about or at most about 42° C.; of at least about 85%per base at a temperature of about or at most about 12° C., at about orat most about 15° C., at about or at most about 20° C., at about or atmost about 25° C., at about or at most about 30° C., at about or at mostabout 35° C., at about or at most about 40° C., at about or at mostabout 42° C., at about or at most about 45° C., at about or at mostabout 50° C.; of at least about 95% per base at a temperature at aboutor at most about 4° C., at about or at most about 8° C., at about or atmost about 12° C., at about or at most about 15° C., at about or at mostabout 20° C., at about or at most about 25° C., at about or at mostabout 30° C., at about or at most about 35° C., at about or at mostabout 40° C., or at about or at most about 42° C.; of at least about 85%per base at a temperature of about or at most about 12° C., at about orat most about 15° C., at about or at most about 20° C., at about or atmost about 25° C., at about or at most about 30° C., at about or at mostabout 35° C., at about or at most about 40° C., at about or at mostabout 42° C., at about or at most about 45° C., at about or at mostabout 50° C.; of at least about 99% per base at a temperature at aboutor at most about 4° C., at about or at most about 8° C., at about or atmost about 12° C., at about or at most about 15° C., at about or at mostabout 20° C., at about or at most about 25° C., at about or at mostabout 30° C., at about or at most about 35° C., at about or at mostabout 40° C., or at about or at most about 42° C.; of at least about 85%per base at a temperature of about or at most about 12° C., at about orat most about 15° C., at about or at most about 20° C., at about or atmost about 25° C., at about or at most about 30° C., at about or at mostabout 35° C., at about or at most about 40° C., at about or at mostabout 42° C., at about or at most about 45° C., at about or at mostabout 50° C.; of at least about 99.5% per base at a temperature at aboutor at most about 4° C., at about or at most about 8° C., at about or atmost about 12° C., at about or at most about 15° C., at about or at mostabout 20° C., at about or at most about 25° C., at about or at mostabout 30° C., at about or at most about 35° C., at about or at mostabout 40° C., or at about or at most about 42° C.; of at least about 85%per base at a temperature of about or at most about 12° C., at about orat most about 15° C., at about or at most about 20° C., at about or atmost about 25° C., at about or at most about 30° C., at about or at mostabout 35° C., at about or at most about 40° C., at about or at mostabout 42° C., at about or at most about 45° C., at about or at mostabout 50° C.; or of about 100% per base at a temperature at about or atmost about 4° C., at about or at most about 8° C., at about or at mostabout 12° C., at about or at most about 15° C., at about or at mostabout 20° C., at about or at most about 25° C., at about or at mostabout 30° C., at about or at most about 35° C., at about or at mostabout 40° C., or at about or at most about 42° C.; of at least about 85%per base at a temperature of about or at most about 12° C., at about orat most about 15° C., at about or at most about 20° C., at about or atmost about 25° C., at about or at most about 30° C., at about or at mostabout 35° C., at about or at most about 40° C., at about or at mostabout 42° C., at about or at most about 45° C., at about or at mostabout 50° C.

In some embodiments, the non-naturally occurring or modified enzyme or amodified polypeptide having reverse transcriptase activity can amplifyor is capable of amplifying a template nucleic acid molecule at aprocessivity of at least about 80% per base at a temperature of at mostabout 35° C., of at least about 85% per base at a temperature of at mostabout 40° C., of at least about 88% per base at a temperature of at mostabout 35° C., of at least about 89% per base at a temperature of at mostabout 40° C., of at least about 90% per base at a temperature of at mostabout 35° C., of at least about 91% per base at a temperature of at mostabout 35° C., of at least about 92% per base at a temperature of at mostabout 40° C., of at least about 93% per base at a temperature of at mostabout 35° C., of at least about 94% per base at a temperature of at mostabout 40° C., of at least about 95% per base at a temperature of at mostabout 35° C., of at least about 96% per base at a temperature of at mostabout 40° C., of at least about 97% per base at a temperature of at mostabout 35° C., of at least about 98% per base at a temperature of at mostabout 40° C., of at least about 99% per base at a temperature of at mostabout 40° C., of at least about 99.5% per base at a temperature of atmost about 40° C., or of about 100% per base at a temperature of at mostabout 40° C.

In some embodiments, the improved enzyme property is selected from atleast one of the following: improved stability (e.g., improvedthermostability), improved specific activity, improved proteinexpression, improved purification, improved processivity, improvedstrand displacement, improved template jumping, improved DNA/RNAaffinity, and improved fidelity. In some embodiments, a non-naturallyoccurring enzyme or a modified enzyme or a modified polypeptide havingreverse transcriptase activity amplifies a template nucleic acidmolecule. In some embodiments, the non-naturally occurring enzyme or themodified enzyme or the modified polypeptide having reverse transcriptaseactivity that amplifies a template nucleic acid molecule has aperformance index greater than about 1, greater than about 2, greaterthan about 3, greater than about 4, greater than about 5, greater thanabout 6, greater than about 7, greater than about 8, greater than about9, greater than about 10, greater than about 15, greater than about 20,greater than about 25, greater than about 30, greater than about 35,greater than about 40, greater than about 45, greater than about 50,greater than about 60, greater than about 70, greater than about 80,greater than about 90, or greater than about 100 for at least one enzymeproperty. In some embodiments, the enzyme property and/or theperformance index is performed at a temperature equal to or lower thanor at most about 50° C., equal to or lower than or at most about 42° C.,equal to or lower than or at most about 40° C., equal to or lower thanor at most about 39° C., equal to or lower than or at most about 38° C.,equal to or lower than or at most about 37° C., equal to or lower thanor at most about 36° C., equal to or lower than or at most about 35° C.,equal to or lower than or at most about 34° C., equal to or lower thanor at most about 33° C., equal to or lower than or at most about 32° C.,equal to or lower than or at most about 31° C., equal to or lower thanor at most about 30° C., equal to or lower than or at most about 29° C.,equal to or lower than or at most about 28° C., equal to or lower thanor at most about 27° C., equal to or lower than or at most about 26° C.,equal to or lower than or at most about 25° C., equal to or lower thanor at most about 23° C., equal to or lower than or at most about 20° C.,equal to or lower than or at most about 15° C., equal to or lower thanor at most about 13° C., equal to or lower than or at most about 12° C.,equal to or lower than or at most about 10° C., equal to or lower thanor at most about 8° C., equal to or lower than or at most about 4° C. Insome embodiments, the non-naturally occurring enzyme or the modifiedenzyme (e.g., modified reverse transcriptase) or the modifiedpolypeptide having reverse transcriptase activity exhibits aprocessivity for a given nucleotide substrate that is at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 37.5%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 90%, at least about 95%, at least about 100%, at leastabout 110%, at least about 125%, at least about 150%, at least about170%, at least about 190%, at least about 200%, at least about 250%, atleast about 500%, at least about 750%, at least about 1000%, at leastabout 5000%, or at least about 10000% higher than the processivity of areference enzyme or a reference polypeptide for the same nucleotidesubstrate. In some embodiments, the non-naturally occurring enzyme is anon-naturally occurring reverse transcriptase enzyme. In someembodiments, the modified enzyme is a modified reverse transcriptase.

The present disclosure relates to processes and/or methods that requireconsiderably less hands-on time, the protocol is much simpler to performand requires a much shorter duration time than other methods used forRNA sequencing and/or liquid biopsy, for example. In some embodiments,the methods and processes of the present disclosure comprises a protocolthat is less than about 2 hours and/or less than about 30 minutes ofhands-on time. In some embodiments, the protocol is less than about 20hours, less than about 15 hours, less than about 12 hours, less thanabout 11 hours, less than about 10 hours, less than about 9 hours, lessthan about 8 hours, less than about 7 hours, less than about 6 hours,less than about 5 hours, less than about 4 hours, less than about 3hours, less than about 2.5 hours, less than about 2 hours, less thanabout 1.5 hours, less than about 1 hour, or less than about 30 minutes.In some embodiments, the hands-on time is less than about 5 hours, lessthan about 4 hours, less than about 3 hours, less than about 2.5 hours,less than about 2 hours, less than about 1.5 hours, less than about 1hour, less than about 50 minutes, less than about 40 minutes, less thanabout 35 minutes, less than about 30 minutes, less than about 25minutes, less than about 20 minutes, or less than about 15 minutes.

In some embodiments, the method for preparing a nucleic acid libraryand/or a complementary cDNA library comprises preparing the library inat most about 1 hour, at most about 2 hours, at most about 3 hours, atmost about 4 hours, at most about 5 hours, at most about 7 hours, atmost about 10 hours, at most about 15 hours, or at most about 20 hours.

In one embodiment, the present disclosure relates to methods andprocesses that enable the discovery of novel markers and mutations forcancer, and/or provides approaches for precision medicine. In someembodiments, the methods and processes disclosed herein provides forhigher sensitivity to capture minor allele in ctDNA of <0.1% (availablecurrent methods have sensitivity >1%). In some embodiments, the methodsand/or processes of the present disclosure comprise a 1-pot (e.g.,single vessel), 1-step protocol, and the library is prepared from asample in an amount of time that is equal to or less than about 2 hours.See FIG. 2.

The present disclosure relates to methods for preparing a modifiedreverse transcriptase, which method comprises at least one of thefollowing steps: (a) subjecting a nucleic acid sequence encoding areverse transcriptase enzyme to random or rational mutagenesis; (b)subjecting a nucleic acid sequence encoding a reverse transcriptaseenzyme to truncation of amino acids; (c) subjecting a nucleic acidsequence encoding a reverse transcriptase enzyme to alterationcomprising an insertion, a deletion or a substitution of an amino acidresidue; and (d) subjecting a nucleic acid sequence encoding a reversetranscriptase enzyme to fusion with a protein or domain. In someembodiments, the nucleic acid sequence obtained in any one of steps (a)to (d) is expressed in a host cell. In some embodiments, the modifiedreverse transcriptase comprises at least one improved enzyme propertyrelative to a wild type or unmodified reverse transcriptase. In someembodiments the nucleic acid sequence is DNA, RNA, or a combination ofRNA and DNA. In some embodiments, the method comprises screening forhost cells expressing modified reverse transcriptase(s). In someembodiments, the method comprises preparing modified reversetranscriptase(s) expressed by the host cell(s). In some embodiments, themethod may comprise purifying the modified reverse transcriptase(s)according to any method including any method disclosed herein. In someembodiments, the method may comprise determining the reversetranscriptase activity, estimating the reverse transcriptase activityfractions, and/or testing the stability and/or robustness of themodified reverse transcriptase(s). In some embodiments, determining thereverse transcriptase activity, estimating the reverse transcriptaseactivity fractions, and/or testing the stability and/or robustness ofthe modified reverse transcriptase(s) are performed or tested using areverse transcriptase activity assay. In some embodiments, the reversetranscriptase activity, the reverse transcriptase active fraction,and/or the stability and/or robustness of the modified reversetranscriptase(s) is increased/improved compared to the unmodified ornaturally occurring reverse transcriptase.

The present disclosure relates to methods for preparing a complementarydeoxyribonucleic acid (cDNA) molecule. In some embodiments, the methodcomprises annealing a primer to a template nucleic acid molecule,thereby generating an annealed template nucleic acid molecule. In someembodiments, the method further comprises mixing, in the presence ofnucleotides, the annealed template nucleic acid molecule, a one or moreacceptor nucleic acid molecules, and a modified reverse transcriptase.In some embodiments, the modified reverse transcriptase generates aplurality of continuous complementary deoxyribonucleic acid molecules.In some embodiments, the plurality of continuous complementarydeoxyribonucleic acid molecules are prepared in at most about 2 hours.In some embodiments, the plurality of continuous complementarydeoxyribonucleic acid molecules is generated by having the modifiedreverse transcriptase reverse transcribe a sequence of the annealedtemplate nucleic acid molecule. In some embodiments, the modifiedreverse transcriptase then migrates to an acceptor nucleic acid molecule(e.g., one or more acceptor nucleic acid molecules). In someembodiments, the reverse transcriptase (e.g., modified reversetranscriptase) is able to reverse transcribe a sequence of the templateand/or the acceptor nucleic acid molecule at a temperature of from about12° C. to about 42° C. In some embodiments, the reverse transcriptase(e.g., modified reverse transcriptase) is able to reverse transcribe asequence of the template and/or the acceptor nucleic acid molecule at atemperature of from about 8° C. to about 50° C. (e.g., about 8° C.,about 15° C., about 20° C., about 25° C., about 30° C., about 35° C.,about 40° C., about 45° C., about 48° C.). In some embodiments, thereverse transcriptase (e.g., modified reverse transcriptase) is able toreverse transcribe a sequence of the template and/or the acceptornucleic acid molecule at a temperature of at most about 4° C., at mostabout 8° C., at most about 15° C., at most about 20° C., at most about25° C., at most about 30° C., at most about 35° C., at most about 40°C., at most about 45° C., or at most about 48° C. In some embodiments,reverse transcription occurs at an error rate of at most about 5%. Insome embodiments, the reverse transcriptase (e.g., modified reversetranscriptase) is capable of reverse transcribing the template and/orthe acceptor nucleic acid molecule at an error rate of at most about45%, at most about 40%, at most about 35%, at most about 30%, at mostabout 25%, at most about 20%, at most about 15%, at most about 10%, atmost about 8%, at most about 7%, at most about 6%, at most about 5%, atmost about 4%, at most about 3%, at most about 2%, or at most about 1%.In some embodiments, the reverse transcriptase (e.g., modified reversetranscriptase) can migrate from the template to the acceptor nucleicacid molecule independently of sequence identity between the templateand the acceptor nucleic acid molecule. In some embodiments, the methodis prepared in a single vessel. In some embodiments, the templatenucleic acid molecule is a fragmented DNA template, a fragmented RNAtemplate, a non-fragmented DNA template, a non-fragmented RNA template,or a combination thereof. In some embodiments, the method furthercomprises adding a tag to a template nucleic acid molecule, therebygenerating a plurality of tagged continuous complementarydeoxyribonucleic acid molecules. In some embodiments, the method furthercomprises performing a polymerase chain reaction amplification reaction,thereby forming one or more amplicons.

The present disclosure relates to methods for preparing a complementarydeoxyribonucleic acid (cDNA) molecule using a modified reversetranscriptase. In some embodiments, the method for preparing a cDNAmolecule is via template jumping. In some embodiments, the modifiedreverse transcriptase has an improved enzyme property compared to anaturally occurring or unmodified or wild type enzyme (e.g., wild typereverse transcriptase). In some embodiments, the method for preparing acDNA molecule comprises: (a) annealing a primer to a template; and (b)mixing, in the presence of nucleotides (e.g., dNTPs), the templateannealed to the primer with a modified reverse transcriptase and anacceptor nucleic acid molecule (e.g., acceptor RNA, DNA, or acombination thereof) under conditions sufficient to generate a cDNAmolecule complementary to the template and/or to the acceptor nucleicacid molecule (FIG. 3). In some embodiments, the enzyme (e.g., modifiedreverse transcriptase) generates a continuous cDNA molecule by migratingfrom the template to the acceptor nucleic acid molecule. In someembodiments, template jumping is independent of sequence identitybetween the template and the acceptor nucleic acid molecule. In someembodiments, step (a) and step (b) are done at the same time. In someembodiments, step (a) comprises step (b) (e.g., step (a) and step (b)are merged into one step). In some embodiments, at least one of step (a)and/or step (b) further comprises addition of a hot start thermostablepolymerase. In some embodiments, the method of the present disclosure isperformed in a single tube. In some embodiments, the method of thepresent disclosure further comprises a polymerase chain reaction (PCR)amplification reaction. In some embodiments, the PCR amplificationreaction is performed in a single tube (e.g., the same one tube fromsteps (a) and (b)). In some embodiments, all the steps of the method ofthe present disclosure are performed in a single tube.

The present disclosure relates to a method for preparing a concatemer ofnucleic acid molecules for sequencing. In some embodiments, the methodcomprises ligating a nucleic acid molecule with a first adaptor. In someembodiments, the method further comprises amplifying the ligated nucleicacid molecule by performing a nucleic acid amplification reaction toform a concatemer. In some embodiments, the amplification reaction isperformed in the absence of a primer. In some embodiments, the methodfurther comprises ligating the concatemer with a second adaptor. In someembodiments, the adaptor(s) (first and/or second adaptor) is/aredesigned to allow recombination or homology based annealing andextension of molecules (e.g., nucleic acid molecules, and/or a template,and/or a primer, and/or an acceptor). In some embodiments, the nucleicacid amplification reaction is polymerase chain reaction (PCR) orisothermal amplification. In some embodiments, the first adaptorcomprises a unique molecular identifier (UMI) sequence. In someembodiments, the first adaptor serves as a primer. In some embodiments,the first adaptor comprises single stranded nucleic acid. In someembodiments, the single stranded nucleic acid comprises single strandedDNA (ssDNA). In some embodiments, the second adaptor comprises doublestranded nucleic acid. In some embodiments, the double stranded nucleicacid comprises double stranded DNA (dsDNA). In some embodiments, thefirst adaptor is different from the second adaptor. In some embodiments,the first adaptor comprises two or more adaptors. In some embodiments,the second adaptor comprises two or more adaptors. In some embodiments,both ends of the nucleic acid molecule comprise an adaptor. In someembodiments, only one end of the nucleic acid molecule comprises anadaptor. In some embodiments, both the 3′ and the 5′ ends of a nucleicacid molecule comprise an adaptor.

The present disclosure relates to methods for preparing a complementarydeoxyribonucleic acid (cDNA) molecule using a modified reversetranscriptase. In some embodiments, the method for preparing a cDNAmolecule is via template jumping. In some embodiments, the modifiedreverse transcriptase has an improved enzyme property compared to anaturally occurring or unmodified or wild type enzyme (e.g., wild typereverse transcriptase). In some embodiments, the method for preparing acDNA molecule comprises mixing, in the presence of nucleotides (e.g.,dNTPs), a primer, a template, a modified reverse transcriptase and anacceptor nucleic acid molecule (e.g., acceptor RNA, DNA, or acombination thereof) under conditions sufficient to generate a cDNAmolecule complementary to the template and/or to the acceptor nucleicacid molecule. In some embodiments, the method comprises addition of ahot start thermostable polymerase (e.g., to the mixing step). In someembodiments, the method of the present disclosure is performed in asingle tube. In some embodiments, the method of the present disclosurefurther comprises a polymerase chain reaction (PCR) amplificationreaction. In some embodiments, the PCR amplification reaction isperformed in a single tube (e.g., the same one tube as the mixing step).In some embodiments, all the steps of the method of the presentdisclosure is performed in a single tube (single vessel).

In some embodiments, the method for preparing a cDNA molecule comprises:(a) annealing one or more primer(s) to a template; and (b) mixing, inthe presence of nucleotides (e.g., dNTPs), the template annealed to oneor more primer(s) with a modified reverse transcriptase and an acceptornucleic acid molecule (e.g., acceptor RNA, DNA, or a combinationthereof) under conditions sufficient to generate a cDNA moleculecomplementary to the template and/or to the acceptor nucleic acidmolecule (FIG. 4). In some embodiments, the method for preparing a cDNAmolecule is via template jumping. In some embodiments, step (a) and step(b) are done at the same time. In some embodiments, step (a) comprisesstep (b) (e.g., step (a) and step (b) are merged into one step). In someembodiments, at least one of step (a) and/or step (b) further comprisesaddition of a hot start thermostable polymerase. In some embodiments,the method of the present disclosure is performed in a single tube. Insome embodiments, the method of the present disclosure further comprisesa polymerase chain reaction (PCR) amplification reaction. In someembodiments, the PCR amplification reaction is performed in a singletube (e.g., the same one tube used in or from steps (a) and (b)). Insome embodiments, all the steps of the method of the present disclosureis performed in a single tube.

In some embodiments, the method for preparing a cDNA molecule comprisesmixing, in the presence of nucleotides (e.g., dNTPs), one or moreprimer(s), a template, a modified reverse transcriptase, and an acceptornucleic acid molecule (e.g., acceptor RNA, DNA, or a combinationthereof) under conditions sufficient to generate a cDNA moleculecomplementary to the template and/or to the acceptor nucleic acidmolecule. In some embodiments, the method for preparing a cDNA moleculeis via template jumping. In some embodiments, the method comprisesaddition of a hot start thermostable polymerase (e.g., to the mixingstep). In some embodiments, the method of the present disclosure isperformed in a single tube. In some embodiments, the method of thepresent disclosure further comprises a polymerase chain reaction (PCR)amplification reaction. In some embodiments, the PCR amplificationreaction is performed in a single tube (e.g., the same one tube as themixing step). In some embodiments, all the steps of the method of thepresent disclosure is performed in a single tube.

The present disclosure relates to methods for preparing a nucleic acidmolecule comprising: mixing, in the presence of nucleotides (e.g.,dNTPs), a fragment or degraded template (e.g., a nucleic acid fragment),a primer, a modified reverse transcriptase, and an acceptor nucleic acidmolecule under conditions sufficient to generate a nucleic acid molecule(FIGS. 5A and 5B). In some embodiments, the acceptor nucleic acidmolecule comprises a modified nucleotide. In some embodiments, theprimer extension stops at the modified nucleotide. In some embodiments,the modified reverse transcriptase comprises at least one improvedenzyme property relative to a wild type, naturally occurring, orunmodified reverse transcriptase. In some embodiments, the primer is anRNA primer. In some embodiments, the primer is an engineered primer(e.g., engineered RNA primer). In some embodiments, the primer has beenoptimized. In some embodiments, the primer is an optimized and/orengineered primer (e.g., optimized and/or engineered RNA primer). Insome embodiments, the primer is RNA R2 primer. In some embodiments, themethod for preparing a nucleic acid molecule is via template jumping. Insome embodiments, the mixing step of the method of the presentdisclosure further comprises addition of a hot start thermostablepolymerase. In some embodiments, the method of the present disclosure isperformed in a single tube. In some embodiments, the method of thepresent disclosure further comprises a polymerase chain reaction (PCR)amplification reaction. In some embodiments, the PCR amplificationreaction is performed in the same single tube. In some embodiments, allthe steps of the method of the present disclosure is performed in asingle tube.

The present disclosure relates to methods for preparing a nucleic acidmolecule comprising: mixing, in the presence of nucleotides (e.g.,dNTPs), a fragment or degraded template (e.g., a nucleic acid fragment),a donor complex, a modified reverse transcriptase, and an acceptornucleic acid molecule under conditions sufficient to generate a nucleicacid molecule (FIGS. 6A and 6B). In some embodiments, the acceptornucleic acid molecule comprises a modified nucleotide. In someembodiments, the primer extension stops at the modified nucleotide. Insome embodiments, the modified reverse transcriptase comprises at leastone improved enzyme property relative to a wild type or naturallyoccurring or unmodified reverse transcriptase. In some embodiments, thedonor complex comprises a template and a primer. In some embodiments,the donor complex is a donor R2 complex. In some embodiments, the donorR2 complex comprises an RNA R2 primer. In some embodiments, the methodfor preparing a nucleic acid molecule is via template jumping. In someembodiments, the mixing step of the method of the present disclosurefurther comprises addition of a hot start thermostable polymerase. Insome embodiments, the method of the present disclosure is performed in asingle tube. In some embodiments, the method of the present disclosurefurther comprises a polymerase chain reaction (PCR) amplificationreaction. In some embodiments, the PCR amplification reaction isperformed in the same single tube (e.g., the same single tube used toprepare a nucleic acid molecule). In some embodiments, all the steps ofthe method of the present disclosure is performed in a single tube.

The present disclosure relates to methods for preparing a complementarydeoxyribonucleic acid (cDNA) library using a modified reversetranscriptase. In some embodiments, the method for preparing a cDNAlibrary uses template jumping. In some embodiments, the modified reversetranscriptase has an improved enzyme property compared to a naturallyoccurring or wild type or unmodified enzyme (e.g., wild type reversetranscriptase). In some embodiments, the method for preparing a cDNAlibrary comprises: (a) annealing a primer or one or more primer(s) to atemplate; and (b) mixing, in the presence of nucleotides (e.g., dNTPs),the template annealed to the primer or the template annealed to one ormore primer(s) with a modified reverse transcriptase and an acceptornucleic acid molecule (e.g., acceptor RNA, DNA, or a combinationthereof) under conditions sufficient to generate a cDNA moleculecomplementary to the template and/or to the acceptor nucleic acidmolecule. In some embodiments, the method for preparing a cDNA librarycomprises mixing, in the presence of nucleotides (e.g., dNTPs), a primeror one or more primer(s), a template, a modified reverse transcriptase,and an acceptor nucleic acid molecule (e.g., acceptor RNA, DNA, or acombination thereof) under conditions sufficient to generate a cDNAmolecule complementary to the template and/or to the acceptor nucleicacid molecule. In some embodiments, the enzyme (e.g., modified reversetranscriptase) generates a continuous cDNA molecule by migrating fromthe template to the acceptor nucleic acid molecule. In some embodiments,template jumping is independent of sequence identity between thetemplate and the acceptor nucleic acid molecule. In some embodiments themethod further comprises amplifying the cDNA molecule to generate a cDNAlibrary. In some embodiments, step (a) and step (b) are done at the sametime. In some embodiments, step (a) comprises step (b) (e.g., step (a)and step (b) are merged into one step). In some embodiments, the mixingstep or at least one of step (a) and/or step (b) further comprisesaddition of a hot start thermostable polymerase. In some embodiments,the method of the present disclosure is performed in a single tube. Insome embodiments, the method of the present disclosure further comprisesa polymerase chain reaction (PCR) amplification reaction. In someembodiments, the PCR amplification reaction is performed in a singletube (e.g., the same one tube used in or from the mixing step, or in orfrom steps (a) and (b)). In some embodiments, all the steps of themethod of the present disclosure is performed in a single tube.

The present disclosure relates to methods for preparing a cDNA and/orDNA library comprising: mixing, in the presence of nucleotides (e.g.,dNTPs), a fragment or degraded template (e.g., a nucleic acid fragment),a primer, a modified reverse transcriptase, and an acceptor nucleic acidmolecule under conditions sufficient to generate a nucleic acid (e.g.,cDNA and/or DNA) molecule. In some embodiments, the acceptor nucleicacid molecule comprises a modified nucleotide. In some embodiments, theprimer extension stops at the modified nucleotide. In some embodiments,the modified reverse transcriptase comprises at least one improvedenzyme property relative to a wild type or unmodified reversetranscriptase. In some embodiments, the primer is an RNA R2 primer. Insome embodiments, the method further comprises amplifying the nucleicacid (e.g., cDNA and/or DNA) molecule to generate a cDNA library. Insome embodiments, the method for preparing a cDNA and/or DNA and/ornucleic acid molecule is via template jumping.

The present disclosure relates to methods for preparing a cDNA and/orDNA library comprising: mixing, in the presence of nucleotides (e.g.,dNTPs), a fragment or degraded template (e.g., a nucleic acid fragment),a donor complex, a modified reverse transcriptase, and an acceptornucleic acid molecule under conditions sufficient to generate a nucleicacid (e.g., cDNA and/or DNA) molecule. In some embodiments, the acceptornucleic acid molecule comprises a modified nucleotide. In someembodiments, the primer extension stops at the modified nucleotide. Insome embodiments, the modified reverse transcriptase comprises at leastone improved enzyme property relative to a wild type or unmodifiedreverse transcriptase. In some embodiments, the donor complex comprisesa template and a primer. In some embodiments, the donor complex is adonor R2 complex. In some embodiments, the donor R2 complex comprises anRNA R2 primer. In some embodiments, the method further comprisesamplifying the nucleic acid (e.g., cDNA and/or DNA) molecule to generatea cDNA and/or DNA library. In some embodiments, the method for preparinga cDNA and/or DNA and/or nucleic acid molecule is via template jumping.

In some embodiments, the method of the present disclosure may comprise adonor complex. In some embodiments, the donor complex comprises atemplate and a primer. In some embodiments, the method of the presentdisclosure may comprise a template. In some embodiments, the template isa fragmented and/or degraded template. In some embodiments, the templateis not fragmented. In some embodiments, the template is RNA, DNA, or acombination of DNA and RNA. In some embodiments, the RNA is mRNA. Insome embodiments, the template is mRNA.

The present disclosure relates to methods for preparing a library forsequencing comprising: (a) obtaining a sample with cell-free nucleicacid from a subject; and (b) adding a modified reverse transcriptaseenzyme, a template (e.g., a nucleic acid template), nucleotides, anacceptor nucleic acid molecule, and one or more primer(s) to the nucleicacid. In some embodiments, the method further comprises conducting anamplification reaction on the cell-free nucleic acid (cf nucleic acid)derived from the sample to produce a plurality of amplicons. In someembodiments, the amplification reaction comprises 35 or feweramplification cycles. In some embodiments, the method comprisesproducing a library for sequencing. In some embodiments, the librarycomprises a plurality of amplicons. In some embodiments, the modifiedreverse transcriptase is capable of template jumping and/or comprises atleast one improved enzyme property relative to a wild type or unmodifiedreverse transcriptase. In some embodiments, the nucleic acid is DNA,RNA, or a combination of RNA and DNA.

The present disclosure relates to a method for preparing a complementarydeoxyribonucleic acid (cDNA) molecule using template jumping, comprisingmixing, in a single tube, a primer or one or more primer(s), a messengerRNA (mRNA) template, nucleotides, a modified reverse transcriptase, anacceptor nucleic acid molecule, and a catalytic metal under conditionssufficient to generate a continuous cDNA molecule. In some embodiments,the continuous cDNA molecule is complementary to the mRNA templateand/or to the acceptor nucleic acid molecule. In some embodiments, themodified reverse transcriptase comprises at least one improved enzymeproperty relative to a wild type or unmodified reverse transcriptase. Insome embodiments, a continuous cDNA molecule is produced. In someembodiments, the modified reverse transcriptase undergoes migration fromthe template to the acceptor nucleic acid molecule.

The present disclosure relates to a method for preparing a library forsequencing comprising mixing, in a single tube, a cell-free nucleicacid, a modified reverse transcriptase enzyme, a template, nucleotides,an acceptor nucleic acid molecule, a catalytic metal, and one or moreprimer(s), under conditions sufficient to generate a library. In someembodiments, the modified reverse transcriptase comprises at least oneimproved enzyme property relative to a wild type or unmodified reversetranscriptase.

In some embodiments, the nucleic acid molecule comprises an unknownnucleic acid sequence. In some embodiments, the template comprises anunknown nucleic acid sequence. In some embodiments, the migration fromthe template to the acceptor nucleic acid molecule is independent ofsequence identity between the template and the acceptor nucleic acidmolecule. In some embodiments, the acceptor nucleic acid moleculecomprises a modified nucleotide that may cause primer extension to stop.In some embodiments, the cell-free nucleic acid is cell-free DNA(cfDNA), circulating tumor DNA (ctDNA), and/or formalin-fixed,paraffin-embedded DNA (FFPE DNA), or combinations thereof.

In some embodiments, a hot start thermostable polymerase may be added toa method of the present disclosure at or prior to any step of the methodand/or at the same time that a mixing step takes place. For example, ahot start thermostable polymerase may be added at the same time that themodified reverse transcriptase is added to the reaction. The hot startthermostable polymerase may be added at the same time that the acceptornucleic acid molecule is added, and/or at the same time that thetemplate, and/or primer, and/or reverse transcriptase, and/ornucleotides is added to the reaction tube. In some embodiments, the hotstart thermostable polymerase is added prior to the start of the PCRreaction. In some embodiments, the hot start thermostable polymerase isadded prior to or at the same time as the RT reaction. In someembodiments, the hot start thermostable polymerase is hot start taqpolymerase. Amplification of target nucleic acids can occur on a bead.In some embodiments, amplification does not occur on a bead.Amplification can be by isothermal amplification, e.g., isothermallinear amplification. In some embodiments, a hot start PCR can beperformed wherein the reaction is heated to 95° C. e.g., for two minutesprior to addition of a polymerase or the polymerase can be kept inactiveuntil a first heating step in cycle 1. Hot start PCR can be used tominimize nonspecific amplification.

In some embodiments, the method of the present disclosure is performedin a single tube. In some embodiments, all the steps of the method ofthe present disclosure is performed in a single tube. In someembodiments, the method (from start to finish) is performed in a singletube. In some embodiments, the same tube used for the RT reaction isused for the PCR amplification reaction.

In some embodiments, the PCR amplification is performed at a temperaturesufficient to inactivate the reverse transcriptase enzyme. In someembodiments, the PCR amplification is performed at a temperaturesufficient to activate the hot start thermostable polymerase.

The present disclosure relates to methods of amplifying a cell-freenucleic acid molecule from a sample. In some embodiments, the sample isa biological sample. In some embodiments, the cell-free nucleic acidmolecule is subjected to nucleic acid amplification comprising a reversetranscriptase (e.g., modified reverse transcriptase). In someembodiments, the cell-free nucleic acid molecule is subjected to nucleicacid amplification comprising a reverse transcriptase (e.g., modifiedreverse transcriptase) under conditions that amplify the nucleic acidmolecule at a specified processivity. In some embodiments theprocessivity is of at least about 80% per base, at least about 81% perbase, at least about 82% per base, at least about 83% per base, at leastabout 84% per base, at least about 85% per base, at least about 86% perbase, at least about 87% per base, at least about 88% per base, at leastabout 89% per base, at least about 90% per base, at least about 91% perbase, at least about 92% per base, at least about 93% per base, at leastabout 94% per base, at least about 95% per base, at least about 96% perbase, at least about 97% per base, at least about 98% per base, at leastabout 99% per base, or at least about 100% per base. In someembodiments, the processivity is performed at a temperature of about orat most about or at least about 12° C., of about or at most about or atleast about 13° C., of about or at most about or at least about 14° C.,of about or at most about or at least about 15° C., of about or at mostabout or at least about 16° C., of about or at most about or at leastabout 17° C., of about or at most about or at least about 18° C., ofabout or at most about or at least about 19° C., of about or at mostabout or at least about 20° C., of about or at most about or at leastabout 21° C., of about or at most about or at least about 22° C., ofabout or at most about or at least about 23° C., of about or at mostabout or at least about 24° C., of about or at most about or at leastabout 25° C., of about or at most about or at least about 26° C., ofabout or at most about or at least about 27° C. of about or at mostabout or at least about 28° C., of about or at most about or at leastabout 29° C., of about or at most about or at least about 30° C., ofabout or at most about or at least about 31° C., of about or at mostabout or at least about 32° C., of about or at most about or at leastabout 33° C., of about or at most about or at least about 34° C., ofabout or at most about or at least about 35° C., of about or at mostabout or at least about 36° C., of about or at most about or at leastabout 37° C., of about or at most about or at least about 38° C., ofabout or at most about or at least about 39° C., of about or at mostabout or at least about 40° C., of about or at most about or at leastabout 45° C., of about or at most about or at least about 50° C., ofabout or at most about or at least about 60° C., of about or at mostabout or at least about 70° C., of about or at most about or at leastabout 80° C., of about or at most about or at least about 8° C. In someembodiments the processivity is of at least about 80% per base, at leastabout 81% per base, at least about 82% per base, at least about 83% perbase, at least about 84% per base, at least about 85% per base, at leastabout 86% per base, at least about 87% per base, at least about 88% perbase, at least about 89% per base, at least about 90% per base, at leastabout 91% per base, at least about 92% per base, at least about 93% perbase, at least about 94% per base, at least about 95% per base, at leastabout 96% per base, at least about 97% per base, at least about 98% perbase, at least about 99% per base, or at least about 100% per base, at atemperature of about or at most about or of at least about 30° C., or ofabout or at most about or of at least about 12° C., of about or at mostabout or of at least about 45° C., of about or at most about or of atleast about 35° C. In some embodiments, the reverse transcriptase is anon-LTR retrotransposon or a modified non-LTR retrotransposon. In someembodiments, the reverse transcriptase is an R2 reverse transcriptase ora modified R2 reverse transcriptase. In some embodiments, the reversetranscriptase is an R2 non-LTR retrotransposon or a modified R2 non-LTRretrotransposon.

The present disclosure relates to methods for preparing a complementarydeoxyribonucleic acid (cDNA) library and/or a DNA library from aplurality of single cells. In some embodiments, the method comprises thesteps of: releasing nucleic acid from each single cell to provide aplurality of individual nucleic acid samples. In some embodiments, thenucleic acid in each individual nucleic acid sample is from a singlecell. In some embodiments, the method further comprises annealing thenucleic acid template to one or more primer(s). In some embodiments, themethod further comprises mixing the nucleic acid template annealed toone or more primer(s) with an acceptor template (or an acceptor nucleicacid molecule) and a modified reverse transcriptase, in the presence ofnucleotides, under conditions effective for producing a cDNA and/or aDNAmolecule. In some embodiments, the modified reverse transcriptase iscapable of template jumping and/or comprises at least one improvedenzyme property relative to a wild type or unmodified reversetranscriptase. In some embodiments, the method further comprisesamplifying the cDNA molecule and/or DNA molecule to generate a cDNAand/or DNA library.

The present disclosure relates to methods of detecting a nucleic acidmolecule. In some embodiments, the method comprises mixing a samplecomprising a nucleic acid molecule with an acceptor template (or anacceptor nucleic acid molecule), a modified reverse transcriptase, aprimer, and nucleotides, under conditions effective for generating anucleic acid molecule. In some embodiments, the modified reversetranscriptase comprises at least one improved enzyme property relativeto a wild type or unmodified reverse transcriptase. In some embodiments,the acceptor template (or an acceptor nucleic acid molecule) comprisesat least one modified nucleotide. In some embodiments, the modifiednucleotide may cause primer extension to stop. In some embodiments, themethod further comprises amplifying the nucleic acid molecule.

The present disclosure relates to methods of detecting, diagnosing,and/or prognosing a disease (e.g., cancer) in a subject comprising: (a)obtaining sequence information of a nucleic acid sample (e.g., acell-free nucleic acid sample) derived from a subject and (b) using thesequence information derived from step (a) to detect circulating tumornucleic acid in the sample. In some embodiments, obtaining sequenceinformation according to step (a) comprises using one or moreadaptor(s). In some embodiments, the one or more adaptor(s) comprises amolecular barcode. An adaptor can comprise one or more endmodifications. An adaptor can comprise one 5′ phosphate. An adaptor cancomprise two 5′ phosphates. An adaptor can comprise one 3′ hydroxyl. Anadaptor can comprise two 3′ hydroxyls. An adaptor can lack a 3′hydroxyl.

In some embodiments, the molecular barcode comprises a randomersequence. In some embodiments, the method is capable of detectingcell-free nucleic acid that is less than or equal to about 0.75%, 0.50%,0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0005%, or 0.00001%, 1%, 1.75%, 1.5%, 1.25%, 2%, 3%,4%, 5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19%,20%, 22%, 25%, 27%, 30%, 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of total cell-free nucleic acid. Insome embodiments, the method is capable of detecting circulating tumornucleic acid that is less than or equal to about 0.75%, 0.50%, 0.25%,0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%,0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%,0.001%, 0.0005%, or 0.00001%, 1%, 1.75%, 1.5%, 1.25%, 2%, 3%, 4%, 5%,6%, 8%, 9%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19%, 20%, 22%,25%, 27%, 30%, 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% of total circulating nucleic acid. In someembodiments, the method is capable of detecting a percentage ofcirculating tumor nucleic acid (ct nucleic acid) that is less than orequal to 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%,0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%,0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005%, or0.00001% of the total cell-free nucleic acid. In some embodiments, thesequence information comprises information related to at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 70, 80, 100,200, or 300 genomic regions. In some embodiments, the sequenceinformation comprises information related to partially all, mostly all,or all genome sequencing. In some embodiments, concentrations as low as50 ng of cfDNA may provide for full genome sequencing.

In some embodiments, the method of the present disclosure may be used todetermine the presence of a disease (e.g., cancer) in a subject. In someembodiments, determining the presence of cancer in a subject comprisesobtaining a sample from a subject and detecting a nucleic acid molecule(e.g., nucleic acid fragment) in the sample according to any of themethods described herein. In some embodiments, determining the presenceof a disease (e.g., cancer) in a subject comprises amplifying and/orsequencing the nucleic acid molecule. In some embodiments, the presenceof a nucleic acid molecule is indicative of cancer. In some embodiments,the presence of a nucleic acid molecule is indicative of a prenatalcondition. In some embodiments, the nucleic acid molecule and/ortemplate comprises an unknown sequence. In some embodiments, the sampleis a biological sample. In some embodiments, the biological samplecomprises circulating tumor DNA. In some embodiments, the biologicalsample comprises a tissue sample.

In some embodiments, the method of the present disclosure comprisesdetecting an amplicon generated by the amplification primers, whereinthe presence of the amplicon determines whether the modified reversetranscriptase is present in the sample.

In some embodiments, the method of the present disclosure comprisesproviding a prenatal diagnosis based on the presence or absence of anucleic acid molecule (e.g., cDNA molecule).

The present disclosure relates to a kit of producing a nucleic acidmolecule (e.g., cDNA molecule) comprising: one or more primer(s),nucleotides, at least one modified reverse transcriptase, a template,and instructions for performing any of the methods disclosed in thepresent disclosure. In some embodiments, a kit can be used for detectingnucleic acid comprising a nucleic acid template (e.g., a DNA template),at least one modified reverse transcriptase, nucleotides, andinstructions for performing any of the methods disclosed in the presentdisclosure. In some embodiments, the modified reverse transcriptasepresent in the kit or to be used with the kit has activity and/or iscapable of template jumping at a temperature equal to or less than aboutor more than about 4° C., 8° C., 12° C., 13° C., 14° C., 15° C., 16° C.,17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C.,26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C.,35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 45° C.,46° C., 47° C., 48° C., 49° C., 50° C., 52° C., 55° C., or 60° C. Insome embodiments, the nucleic acid and/or the template (e.g., nucleicacid template, DNA, or RNA) is present at a concentration as low asabout 50 femtomolar, as low as about 60 femtomolar, as low as about 70femtomolar, as low as about 75 femtomolar, as low as about 80femtomolar, as low as about 90 femtomolar, as low as about 100femtomolar, as low as about 120 femtomolar, as low as about 150femtomolar, as low as about 200 femtomolar, as low as about 250femtomolar, as low as about 300 femtomolar, as low as about 350femtomolar, as low as about 400 femtomolar, as low as about 500femtomolar, as low as about 550 femtomolar, as low as about 600femtomolar, as low as about 700 femtomolar, or as low as about 800femtomolar. In some embodiments, a kit may comprise one or moreprimer(s), and/or a template annealed to a primer. The presentdisclosure also relates to a kit of producing modified enzymes, modifiedreverse transcriptases, or modified polypeptides. In some embodiments,the kit includes a PCR step and/or components to use for PCR.

In some embodiments, the present disclosure relates to a kit fordetecting nucleic acid comprising a template, at least one modifiedreverse transcriptase, nucleotides, and instructions to perform themethod of the present disclosure. In some embodiments, the nucleic acidis present at a concentration of at least about 50 femtomolar, at leastabout 20 femtomolar, at least about 100 femtomolar, or greater thanabout 1000 femtomolar.

The present disclosure relates to any method disclosed herein whereinthe methods may further comprise detecting at least one amplicongenerated by the amplification primers. In some embodiments, thepresence of at least one amplicon indicates the presence of at least onemodified reverse transcriptase in a sample.

In some embodiments, any of the methods of the present disclosure doesnot comprise a purification step. In some embodiments, any of themethods of the present disclosure comprises at least one purificationstep. In some embodiments, any of the methods of the present disclosurecomprises at least two purification steps. In some embodiments, any ofthe methods of the present disclosure comprises at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten, at least fifteen, or at least twentypurification steps.

The present disclosure relates to a method for preparing a library forsequencing.

In some embodiments, the modified reverse transcriptase is a modifiednon-retroviral reverse transcriptase. In some embodiments, the modifiedreverse transcriptase is a modified non-LTR retrotransposon. In someembodiments, the modified reverse transcriptase is a modified R2 reversetranscriptase.

In some embodiments, the variants or modified enzymes or non-naturallyoccurring enzymes or modified polypeptides have/has improved enzymeproperty compared to the unmodified, wild type or naturally occurringenzyme or polypeptide. In some embodiments, the improved enzyme propertyis selected from at least one of the following: increased stability(e.g., increased thermostability), increased specific activity,increased protein expression, improved purification, improvedprocessivity, improved strand displacement, increased template jumping,and improved fidelity. In some embodiments, the term stability mayinclude, but it is not limited to, thermal stability, storage stability,and pH stability. In some embodiments, specific activity is ameasurement of the enzymatic activity (in units) of the protein orenzyme relative to the total amount of protein or enzyme used in areaction. In some embodiments, specific activity is measured based onthe ability of the enzyme to produce cDNA molecule. In some embodiments,the specific activity is measured in U/mg protein determined based on aprimer extension reaction. In some embodiments, the altered or improvedproperty may be characterized by a Performance Index (PI), where the PIis a ratio of performance of the variant, the modified enzyme, or thenon-naturally occurring enzyme compared to the wild-type or compared toa naturally occurring enzyme or protein. The term “performance index(PI)” may refer to the ratio of performance of a variant polypeptide toa parent polypeptide or of a modified enzyme to an unmodified enzyme(e.g., reverse transcriptase) or of a non-naturally occurring enzyme toa naturally-occurring enzyme for a specified performance characteristic.In some embodiments, the specified performance or enzyme propertycharacteristic may include, but is not limited to, stability (e.g.,thermostability), specific activity, protein expression, purification,processivity, strand displacement, end-to-end template jumping, and/orfidelity. In some embodiments, the PI is greater than about 0.5, whilein other embodiments, the PI is about 1 or is greater than about 1. Insome embodiments, the variant polypeptide, modified enzyme (e.g.,modified reverse transcriptase), or the non-naturally occurring enzymecomprises a modification at one or more amino acid positions. In someembodiments, the modified enzyme or the non-naturally occurring enzymehas a performance index (PI) that is equal to or greater than about 0.1,equal to or greater than about 0.2, equal to or greater than about 0.3,equal to or greater than about 0.4, equal to or greater than about 0.5,equal to or greater than about 0.6, equal to or greater than about 0.7,equal to or greater than about 0.8, equal to or greater than about 0.9,equal to or greater than about 1, equal to or greater than about 1.2,equal to or greater than about 1.5, equal to or greater than about 2,equal to or greater than about 2.5, equal to or greater than about 3,equal to or greater than about 3.5, equal to or greater than about 4,equal to or greater than about 4.5, equal to or greater than about 5,equal to or greater than about 5.5, equal to or greater than about 6,equal to or greater than about 6.5, equal to or greater than about 7,equal to or greater than about 8, equal to or greater than about 9,equal to or greater than about 10, equal to or greater than about 50,equal to or greater than about 75, equal to or greater than about 100,equal to or greater than about 500, equal to or greater than about 1000.In some embodiments, the variant or modified enzyme has a performanceindex (PI) from about 0.1 to about 1, from about 0.5 to about 1, fromabout 0.1 to about 2, from about 1 to about 2, from about 0.5 to about2, from about 0.5 to about 10, from about 1 to about 10, from about 0.1to about 10, from about 1 to about 5, from about 0.5 to about 5, fromabout 0.5 to about 20, from about 0.3 to about 20, from about 5 to about10, from about 1.5 to about 10, from about 1.5 to about 50, from about 1to about 50, from about 1.5 to about 100, from about 1.5 to about 75,from about 4 to about 10, from 3 to about 10, from about 3 to about 25,from about 3 to about 50, from about 2 to about 20, from about 2 toabout 100, from about 2 to about 1000, from about 1 to about 1000. Insome embodiments, the performance index is determined for proteinexpression. In some embodiments, the performance index is determined forat least one characteristic that improves enzyme property. In someembodiments, the performance index is determined for purification. Insome embodiments, the performance index is determined for stability(e.g., thermostability). In some embodiments, the performance index isdetermined for specific activity. In some embodiments, the performanceindex is determined for processivity. In some embodiments, theperformance index is determined for strand displacement. In someembodiments, the performance index is determined for template jumping.In some embodiments, the performance index is determined for fidelity.In some embodiments, the characteristic that improves enzyme property isselected from the group consisting of increased thermal stability,increased specific activity, and increased protein expression. In someembodiments, the performance index is performed at 30° C. In someembodiments, the enzyme property is analyzed at 30° C. In someembodiments, the enzyme property, stability (e.g., thermostability),specific activity, protein expression, purification, processivity,strand displacement, template jumping, and/or fidelity is performed at30° C. In some embodiments, the performance index for measuring enzymeproperty, is performed at a specific temperature. In some embodiments,the temperature is from about 25° C. to about 42° C. In someembodiments, the temperature is from about 8° C. to about 50° C. In someembodiments, the performance index for measuring enzyme property may becarried out at a temperature ranging from about from about 8° C. toabout 50° C., from about 12° C. to about 42° C., 25° C. to about 42° C.,from about 25° C. to about 40° C., from about 28° C. to about 38° C.,from about 30° C. to about 38° C., from about 35° C. to about 37° C.,from about 27° C. to about 38° C., from about 27° C. to about 37° C.,from about 26° C. to about 42° C., from about 25° C. to about 38° C.,from about 27° C. to about 38° C., from about 29° C. to about 38° C.,from about 29° C. to about 32° C. In some embodiments, the performanceindex for measuring enzyme property may be carried out at a temperaturethat is equal to or lower than about 8° C., equal to or lower than about12° C., equal to or lower than about 20° C., equal to or lower thanabout 4° C., equal to or lower than about 55° C., equal to or lower thanabout 37° C., equal to or lower than about 25° C., equal to or lowerthan about 28° C., equal to or lower than about 30° C., equal to orlower than about 32° C., equal to or lower than about 34° C., equal toor lower than about 35° C., equal to or lower than about 36° C., equalto or lower than about 33° C., equal to or lower than about 31° C.,equal to or lower than about 60° C., equal to or lower than about 38°C., equal to or lower than about 39° C., equal to or lower than about40° C., equal to or lower than about 41° C., equal to or lower thanabout 42° C., equal to or lower than about 50° C. In some embodiments,the temperature may range from about 25° C. to about 80° C.

In some embodiments, the specific activity of the modified enzyme isfrom about 5 units/mg to about 140,000 units/mg, from about 5 units/mgto about 125,000 units/mg, from about 50 units/mg to about 100,000units/mg, from about 100 units/mg to about 100,000 units/mg, from about250 units/mg to about 100,000 units/mg, from about 500 units/mg to about100,000 units/mg, from about 1000 units/mg to about 100,000 units/mg,from about 5000 units/mg to about 100,000 units/mg, from about 10,000units/mg to about 100,000 units/mg, from about 25,000 units/mg to about75,000 units/mg. In some embodiments, the ranges of specific activitiesinclude a specific activity of from about 20,000 units/mg to about140,000 units/mg, a specific activity from about 20,000 units/mg toabout 130,000 units/mg, a specific activity from about 20,000 units/mgto about 120,000 units/mg, a specific activity from about 20,000units/mg to about 110,000 units/mg, a specific activity from about20,000 units/mg to about 100,000 units/mg, a specific activity fromabout 20,000 units/mg to about 90,000 units/mg, a specific activity fromabout 25,000 units/mg to about 140,000 units/mg, a specific activityfrom about 25,000 units/mg to about 130,000 units/mg, a specificactivity from about 25,000 units/mg to about 120,000 units/mg, aspecific activity from about 25,000 units/mg to about 110,000 units/mg,a specific activity from about 25,000 units/mg to about 100,000units/mg, and a specific activity from about 25,000 units/mg to about90,000 units/mg. In some embodiments, the lower end of the specificactivity range may vary from 30,000, 35,000, 40,000, 45,000, 50,000,55,000, 60,000, 65,000, 70,000, 75,000, and 80,000 units/mg. In someembodiments, the upper end of the range may vary from 150,000, 140,000,130,000, 120,000, 110,000, 100,000, and 90,000 units/mg.

In some embodiments, the sample is a biological sample. In someembodiments, the biological sample comprises a circulating tumor DNA. Insome embodiments, the biological sample comprises a tissue sample. Insome embodiments, the nucleic acid is from a sample. In someembodiments, the sample is a liquid biopsy sample. In some embodiments,a sample may be an RNA sample. In some embodiments, an RNA sample may beused for various purposes, including but not limited to PCR, ligation,transcriptome analysis, microarray analysis, northern analysis, and cDNAlibrary construction. In some embodiments, the present disclosure isdirected to methods for amplifying cDNA libraries from low quantities ofcells and/or single cells in suitable quantity and quality fortranscriptome analysis through, for example, sequencing or microarrayanalysis.

In some embodiments, the nucleic acid and/or a template is of an unknownsequence. In some embodiments, the nucleic acid and/or a template isRNA, DNA, or a combination of RNA and DNA. In some embodiments, the RNAis mRNA. In some embodiments, the mRNA comprises internal priming. Insome embodiments, the nucleic acid may be a fragmented nucleic acidand/or a degraded nucleic acid. In some embodiments, the template may bea fragmented template and/or a degraded template. In some embodiments,the nucleic acid may be a non-fragmented nucleic acid and/or anon-degraded nucleic acid. In some embodiments, the template may be anon-fragmented template and/or a non-degraded template. In someembodiments, the nucleic acid and/or template is indicative of adisease. In some embodiments, the nucleic acid and/or template isindicative of cancer. In some embodiments, the nucleic acid is equal toor less than about 0.01 micromolar. In some embodiments, the nucleicacid is between about 0.1 nM to about 100 nM. In some embodiments, thenucleic acid is equal to or less than about 500 femtomolar.

In some embodiments, the RNA is obtained from a source selected from thegroup consisting of single cells, cultured cells, tissues, RNAtranscription-based amplified RNA (such as TTR-amplified RNA or otherDNA-dependent RNA polymerase transcribed RNA), RNA-promoter-driventranscribed RNA, aRNA, aRNA-amplified RNA, single-cell mRNA library,isolated mRNA, RNA contained within cells, and combinations of RNAsources. In some embodiments, the RNA is prepared from a plurality offixed cells, wherein said fixed cells are protected from RNA degradationand also subjected to permeabilisation for enzyme penetration. In someembodiments, the fixed cells are obtained from fixative-treated culturalcells, frozen fresh tissues, fixative-treated fresh tissues orparaffin-embedded tissues on slides.

In some embodiments, the RNA molecule can be the product of in vitrosynthesis or can have been isolated from cells or tissues (Ausubel, et.al., Short Protocols in Molecular Biology, 3rd ed., Wiley, 1995). Cellsand tissues suitable for use in obtaining RNA useful in the practice ofthe present disclosure may include both animal cells and plant cells. Insome embodiments, the cells include mammalian cells and insect cells.RNA may also be isolated from prokaryotic cells such as bacteria.

In some embodiments, the template is RNA, DNA, or a combination of RNAand DNA. In some embodiments, the template may be a fragmented templateand/or a degraded template. In some embodiments, the template is notdegraded and/or fragmented. In some embodiments, the RNA is mRNA. Insome embodiments, the template is an RNA template. In some embodiments,the template is a DNA template. In some embodiments, the template is aDNA and/or RNA template. In some embodiments, the template is a mixtureof DNA and RNA. In some embodiments, the RNA comprises any type of RNA(e.g., one or more of rRNA, tRNA, mRNA, and/or snRNA). In someembodiments the RNA comprises a mixture of at least one type of RNA. Insome embodiments, the DNA can comprise a mixture of, or at least one of,genomic DNA or nuclear DNA, mitochondrial DNA, Y-line DNA, autosomalDNA, ribosomal DNA, or a combination thereof. In some embodiments, thetemplate is a polymer of any length. In some embodiments, the templateis from about 20 bases to about 100 bases, from about 30 bases to about500 bases, from about 30 bases to about 1000 bases, from about 50 basesto about 300 bases, about 100 bases to about 600 bases, about 200 basesto about 800 bases, about 200 bases to about 600 bases, about 100 basesto about 2000 bases, about 100 bases and about 2500 bases, about 200bases to about 5000 bases, about 200 bases to about 1000 bases, about200 to about 10000 bases. In some embodiments, the template is at leastabout 10 bases, at least about 20 bases, at least about 30 bases, atleast about 40 bases, at least about 50 bases, at least about 60 bases,at least about 70 bases, at least about 80 bases, at least about 90bases, at least about 100 bases, at least about 150 bases, at leastabout 200 bases, at least about 250 bases, at least about 300 bases, atleast about 350 bases, at least about 400 bases, at least about 450bases, at least about 500 bases, at least about 550 bases, at leastabout 600 bases, at least about 650 bases, at least about 700 bases, atleast about 750 bases, at least about 800 bases, at least about 850bases, at least about 900 bases, at least about 950 bases, at leastabout 1000 bases, at least about 1100 bases, at least about 1200 bases,at least about 1300 bases, at least about 1400 bases, at least about1500 bases, at least about 1700 bases, at least about 2000 bases, atleast about 2200 bases, at least about 2500 bases, at least about 2700bases, at least about 3000, at least about 3500 bases, at least about4000 bases, at least about 4500 bases, at least about 5000 bases, atleast about 10,000 bases, or at least about 50,000 bases. In someembodiments, the template is about or at least about or at most about 10bases, about or at least about or at most about 20 bases, about or atleast about or at most about 30 bases, about or at least about or atmost about 40 bases, about or at least about or at most about 50 bases,about or at least about or at most about 60 bases, about or at leastabout or at most about 70 bases, about or at least about or at mostabout 80 bases, about or at least about or at most about 90 bases, aboutor at least about or at most about 100 bases, about or at least about orat most about 150 bases, about or at least about or at most about 200bases, about or at least about or at most about 250 bases, about or atleast about or at most about 300 bases, about or at least about or atmost about 350 bases, about or at least about or at most about 400bases, about or at least about or at most about 450 bases, about or atleast about or at most about 500 bases, about or at least about or atmost about 550 bases, about or at least about or at most about 600bases, about or at least about or at most about 650 bases, about or atleast about or at most about 700 bases, about or at least about or atmost about 750 bases, about or at least about or at most about 800bases, about or at least about or at most about 850 bases, about or atleast about or at most about 900 bases, about or at least about or atmost about 950 bases, about or at least about or at most about 1000bases, about or at least about or at most about 1100 bases, about or atleast about or at most about 1200 bases, about or at least about or atmost about 1300 bases, about or at least about or at most about 1400bases, about or at least about or at most about 1500 bases, about or atleast about or at most about 1700 bases, about or at least about or atmost about 2000 bases, about or at least about or at most about 2200bases, about or at least about or at most about 2500 bases, about or atleast about or at most about 2700 bases, about or at least about or atmost about 3000, about or at least about or at most about 3500 bases,about or at least about or at most about 4000 bases, about or at leastabout or at most about 4500 bases, about or at least about or at mostabout 5000 bases, about or at least about or at most about 10,000 bases,or about or at least about or at most about 50,000 bases. In someembodiments, the template DNA may be a double-stranded DNA template(dsDNA template) or a single-stranded DNA template (ssDNA template). Insome embodiments, the template RNA may be a double-stranded RNA template(dsRNA template) or a single-stranded RNA template (ssRNA template).

In some embodiments, the template is from a single cell. In someembodiments, the template is from a plurality of cells. In someembodiments, the template comprises low copy number DNA, or RNA, or acombination of DNA and/or RNA. In some embodiments, low copy numberrefers to samples that contain equal to or less than about 250 picograms(e.g. 100 picograms) of for example the template and/or DNA and/or RNAand/or a mixture of DNA and RNA. In some embodiments, the RNA cancomprise at least one of messenger RNA (mRNA), transfer RNA,transfer-messenger RNA, ribosomal RNA, antisense RNA, small nuclear RNA(snRNA), small nucleolar RNA (snoRNA), micro-RNA (miRNA), smallinterfering RNA (siRNA), or any combination thereof. In someembodiments, the template is from a sample. In some embodiments, thetotal amount of template is the total amount of template in a sample. Insome embodiments, the total amount of template is the total amount oftemplate in a reaction mixture. In some embodiments, the total amount oftemplate is the total amount of template in one pot (e.g., singlevessel). In some embodiments, the total amount of the template is fromabout 1 femtomolar (fM) to about 100 micromolar, from about 40femtomolar to about 0.01 micromolar, from about 50 femtomolar to about500 femtomolar, from about 50 femtomolar to about 0.01 micromolar, fromabout 50 femtomolar to about 0.1 micromolar, from about 50 femtomolar toabout 500 picomolar, from about 50 femtomolar to about 500 nanomolar,from about 50 femtomolar to about 500 micromolar, from about 50femtomolar to about 1 picomolar, from about 40 femtomolar to about 1nanomolar, from about 1 femtomolar to about 1 picomolar, from about0.0001 micromolar to about 0.01 micromolar, from about 0.0001 micromolarto about 0.1 micromolar, or from about 0.1 nM to about 100 nM. In someembodiments, the total about of template is equal to or at least aboutor lower than about 1000 micromolar, equal to or at least about or lowerthan about 500 micromolar, equal to or at least about or lower thanabout 250 micromolar, equal to or at least about or lower than about 100micromolar, equal to or at least about or lower than about 50micromolar, equal to or at least about or lower than about 25micromolar, equal to or at least about or lower than about 10micromolar, equal to or at least about or lower than about 1 micromolar,equal to or at least about or lower than about 0.1 micromolar, equal toor at least about or lower than about 0.01 micromolar, equal to or atleast about or lower than about 0.001 micromolar, equal to or at leastabout or lower than about 0.0001 micromolar, equal to or at least aboutor lower than about 2000 nanomolar, equal to or at least about or lowerthan about 500 nanomolar, equal to or at least about or lower than about250 nanomolar, equal to or at least about or lower than about 200nanomolar, equal to or at least about or lower than about 50 nanomolar,equal to or at least about or lower than about 25 nanomolar, equal to orat least about or lower than about 20 nanomolar, equal to or at leastabout or lower than about 2 nanomolar, equal to or at least about orlower than about 0.2 nanomolar, equal to or at least about or lower thanabout 0.01 nanomolar, equal to or at least about or lower than about0.001 nanomolar, equal to or at least about or lower than about 0.0001nanomolar, equal to or at least about or lower than about 3000picomolar, equal to or at least about or lower than about 500 picomolar,equal to or at least about or lower than about 250 picomolar, equal toor at least about or lower than about 300 picomolar, equal to or atleast about or lower than about 50 picomolar, equal to or at least aboutor lower than about 25 picomolar, equal to or at least about or lowerthan about 30 picomolar, equal to or at least about or lower than about3 picomolar, equal to or at least about or lower than about 0.3picomolar, equal to or at least about or lower than about 0.01picomolar, equal to or at least about or lower than about 0.001picomolar, equal to or at least about or lower than about 0.0001picomolar, equal to or at least about or lower than about 5000femtomolar, equal to or at least about or lower than about 500femtomolar, equal to or at least about or lower than about 250femtomolar, equal to or at least about or lower than about 50femtomolar, equal to or at least about or lower than about 25femtomolar, equal to or at least about or lower than about 10femtomolar, equal to or at least about or lower than about 1 femtomolar,equal to or at least about or lower than about 0.1 femtomolar, equal toor at least about or lower than about 0.01 femtomolar, equal to or atleast about or lower than about 0.001 femtomolar, equal to or at leastabout or lower than about 0.0001 femtomolar.

In some embodiments, the template may be present in any nucleic acidsample of interest, including but not limited to, a nucleic acid sampleisolated from a single cell, a plurality of cells (e.g., culturedcells), a tissue, an organ, or an organism (e.g., bacteria, yeast, orthe like). In some embodiments, the nucleic acid sample is isolated froma cell(s), tissue, organ, and/or the like of a mammal (e.g., a human, arodent (e.g., a mouse), or any other mammal of interest). In someembodiments, the nucleic acid sample is isolated from a source otherthan a mammal, such as bacteria, yeast, insects (e.g., drosophila),amphibians (e.g., frogs (e.g., Xenopus)), viruses, plants, or any othernon-mammalian nucleic acid sample source.

In some embodiments, the template is optimized. In some embodiments, theacceptor template or acceptor nucleic acid molecule comprises at leastone modified nucleotide. In some embodiments, the acceptor template oracceptor nucleic acid molecule is engineered to improve template jumpingand/or conversion efficiency. In some embodiments, the acceptor templateor acceptor nucleic acid molecule is optimized at the 3′-end. In someembodiments, the optimization prevents secondary structure formationand/or nucleotide composition.

In some embodiments, the methods disclosed in the present disclosure mayfurther comprise optimization of the template (e.g. donor template). Insome embodiments, optimization of the template comprises contacting thetemplate (e.g. RNA) with an agent capable of removing the 5′ capstructure off the template (e.g., mRNA). In some embodiments, theremoval of the cap structure is performed under conditions permittingthe removal of the cap structure by the agent. In some embodiments, themethods disclosed in the present disclosure further includedephosphorylation of for example, the decapped template. In someembodiments, the method further includes adding a dephosphorylatingagent to the decapped template under conditions permittingdephosphorylation.

In some embodiments, any method of the present disclosure may furthercomprise optimization of the template. In some embodiments, optimizationof the template comprises: contacting a sample comprising a templatewith an agent that removes a 5′ cap structure of the template, underconditions permitting the removal of the cap structure by the agent. Insome embodiments, the optimization of the template may further compriseadding a dephosphorylating agent under conditions permitting thedephosphorylation of the decapped template by the agent. In someembodiments, the template (e.g. RNA molecule) is dephosphorylated aftersynthesis or isolation. In some embodiments, the dephosphorylation isachieved by treatment of the nucleic acid (e.g., RNA) molecule withalkaline phosphatase. In some embodiments, the isolated donor template,such as RNA or mRNA, is decapped and dephosphorylated after isolation.Methods of decapping nucleic acids (e.g., RNAs) include both enzymaticmethods (such as by using a pyrophosphatase such as tobaccopyrophosphatase) and chemical methods (such as periodate oxidation andbeta elimination). Methods for dephosphorylation of nucleic acid (e.g.,RNA) may use alkaline phosphatase. In some embodiments, the isolatedmRNA is decapped (using tobacco acid pyrophosphatase, for example) anddephosphorylated (e.g., by using alkaline phosphatase). In someembodiments, the removal of the RNA cap structure is by either enzymatictreatment of the mRNA with a pyrophosphatase or chemical decapping(e.g., by periodate oxidation and beta elimination). In someembodiments, the mRNA is modified with a tag.

In some embodiments, any of the methods of the present disclosure iscarried out in one-pot (e.g., single vessel). In some embodiments, thereactions are carried out in a one-pot (e.g., single vessel). In someembodiments, the reaction is a one-pot (e.g., single vessel) reaction.

In some embodiments, template jumping is dependent on the concentrationof the acceptor nucleic acid molecule.

In some embodiments, the modified enzyme (e.g., modified reversetranscriptase), modified reverse transcriptase, non-naturally occurringenzyme, modified polypeptide having reverse transcriptase activitycomprises at least one modification relative to the wild type,unmodified counterpart, or naturally occurring enzyme. In someembodiments, the modified non-LTR retrotransposon comprises at least onemodification of a wild-type or unmodified non-LTR retrotransposon. Insome embodiments, the modified R2 reverse transcriptase comprises atleast one modification of a wild-type or unmodified R2 reversetranscriptase. In some embodiments, the modified reverse transcriptasecomprises at least one modification of a wild-type or unmodified reversetranscriptase. In some embodiments, the modified polypeptide havingreverse transcriptase activity comprises at least one modification of awild-type or unmodified polypeptide having reverse transcriptaseactivity. In some embodiments, the modification comprises at least onetruncation (e.g., N-terminal truncation, C-terminal truncation, and/orN- and C-terminal truncations). In some embodiments, the modificationcomprise(s) site-specific incorporation, and/or addition, and/ordeletion, and/or substitution of amino acid(s) at positions of interest.In some embodiments, the modification enhances the biological propertiesof the modified enzyme or modified polypeptide relative to the wild-typeor unmodified enzyme or polypeptide. In some embodiments, themodification improves at least one enzyme property of the modifiedenzyme or polypeptide relative to the wild-type or unmodified enzyme orpolypeptide. In some embodiments, the modification(s) serve as a pointof attachment for, e.g., labels and protein half-life extension agents,and for purposes of affixing the variants to the surface of a solidsupport. In some embodiments, the present disclosure is related tomethods of producing cells capable of producing the modified enzymes(e.g., modified reverse transcriptase) or modified polypeptides, and ofproducing vectors containing DNA or RNA encoding the modified enzymes(e.g., modified reverse transcriptase) or modified polypeptides. In someembodiments, the truncation is based on a two-step process. In someembodiments, the first step for selecting a truncation includesanalyzing the domains and motifs structure(s) and function(s) of a classof enzymes, or proteins, or polypeptides. In some embodiments, theenzymes, or proteins, or polypeptides are non-LTR retrotransposons,reverse transcriptases, R2 reverse transcriptase, LTR retrotransposons,R2 non-LTR retrotransposons, or any combination thereof. In someembodiments, the enzymes, or proteins, or polypeptides are fromdifferent organisms. In some embodiments, all the domains of theenzymes, or proteins, or polypeptides are present. In some embodiments,all the domains are present to ensure reverse transcriptase activity. Insome embodiments, all the domains are present to ensure the uniqueproperties essential for the present disclosure. In some embodiments,the domains responsible for reverse transcriptase activity are notmodified. In some embodiments, the R2 domain does not comprisemodifications. In some embodiments, the R2 domain may comprisemodifications. In some embodiments, the truncated variants showexpression level. In some embodiments, the truncated variants that showpromising expression level are further subject to small adjustment(s) inthe sequence (step two). In some embodiments, the small adjustment(s) inthe sequence include deletion, insertion, and/or substitution of aminoacid(s). In some embodiments, the deletion, insertion, and/orsubstitution of amino acid(s) may include one or several amino acid(s).In some embodiments, the deletion, insertion, and/or substitution ofamino acid(s) further optimize expression and/or stability (e.g.,thermostability).

In some embodiments, the modified enzyme (e.g., modified reversetranscriptase), modified reverse transcriptase, or modified polypeptideshas an N-terminal truncation, a C-terminal truncation, or both, relativeto the wild type or unmodified enzyme (e.g., wild-type reversetranscriptase) or wild-type or unmodified polypeptide. In someembodiments, the polymerase comprises an N-terminal truncation, aC-terminal truncation, or both. In some embodiments, the modifiedreverse transcriptase comprises N-terminal truncation, C-terminaltruncation, or a combination of N-terminal and C-terminal truncation(s).In some embodiments, the modified enzyme comprises N-terminaltruncation, C-terminal truncation, or a combination of N-terminal andC-terminal truncation(s). In some embodiments, the modified polypeptidecomprises N-terminal truncation, C-terminal truncation, or a combinationof N-terminal and C-terminal truncation(s). In some embodiments, thetruncation comprises the sequence MAHHHHHHVGTVGTGGGSGGASTAL. In someembodiments, the modified reverse transcriptase, modified enzyme,modified polypeptide, modified non-LTR retrotransposon, or modified R2reverse transcriptase comprises a truncation of less than about 100amino acid residues. In some embodiments, the modified reversetranscriptase, modified enzyme, modified polypeptide, modified non-LTRretrotransposon, or modified R2 reverse transcriptase comprises at leastone of: (a) an amino-terminal truncation of less than about 400 aminoacid residues and (b) a carboxyl-terminal truncation of less than about400 amino acid residues. In some embodiments, the modified reversetranscriptase, modified enzyme, modified polypeptide, modified non-LTRretrotransposon, or modified R2 reverse transcriptase lacks up to: about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 30, about 50, about 75, about 100, about 120,about 150, about 175, about 200, about 220, about 250, about 275, about280, about 290, about 300, about 325, about 350, about 375, about 380,about 390, about 400, or about 450 amino acids from the N-terminus,C-terminus, or both. In some embodiments, the modified reversetranscriptase, modified enzyme, modified polypeptide, modified non-LTRretrotransposon, or modified R2 reverse transcriptase may alternately oradditionally have one or more internal deletions of up to: about 1,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, or about 25 amino acids, about 30, about 50, about 75, about100, about 120, about 150, about 175, about 200, about 220, about 250,about 275, about 280, about 290, about 300, about 325, about 350, about375, about 380, about 390, or a total of about 450 amino acids. In someembodiments, the N-terminal truncation, C-terminal truncation, or both,may comprise deletions from about 1 to about 50 amino acids, from about1 to about 25, from about 1 to about 70, from about 10 to about 50, fromabout 20 to about 30, from about 15 to about 100, from about 1 to about150, from about 15 to about 60, from about 15 to about 40, from about 1to about 10, from about 10 to 35, from about 50 to about 100, from about20 to about 150, from about 200 to about 350, from about 25 to about350, from about 150 to about 400, from about 50 to about 400, from about50 to about 450, from about 200 to about 400, or from about 50 to about350, or from about 50 to about 400 amino acids. In some embodiments, theN-terminal truncation removes at least about 5, at least about 10, atleast about 15, at least about 20, at least about 25, at least about 30,at least about 35, at least about 40, at least about 50, at least about60, at least about 65, at least about 70, at least about 75, at leastabout 80, at least about 90, at least about 95, at least about 100, atleast about 120, at least about 130, at least about 140, at least about150, at least about 175, at least about 200, at least about 220, atleast about 250, at least about 275, at least about 300, at least about325, at least about 350, at least about 375, or at least about 400 aminoacids. In some embodiments, the C-terminal truncation removes at leastabout 5, at least about 10, at least about 15, at least about 20, atleast about 25, at least about 30, at least about 35, at least about 40,at least about 50, at least about 60, at least about 65, at least about70, at least about 75, at least about 80, at least about 90, at leastabout 95, at least about 100, at least about 120, at least about 130, atleast about 140, at least about 150, at least about 175, at least about200, at least about 220, at least about 250, at least about 275, atleast about 300, at least about 325, at least about 350, at least about375, or at least about 400 amino acids. In some embodiments, theN-terminal truncation lacks about 5, about 10, about 15, about 20, about25, about 30, about 35, about 40, about 50, about 60, about 65, about70, about 75, about 80, about 90, about 95, about 100, about 120, about130, about 140, about 150, about 175, about 200, about 220, about 250,about 275, about 300, about 325, about 350, about 375, or about 400amino acids. In some embodiments, the C-terminal truncation lacks about5, about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 50, about 60, about 65, about 70, about 75, about 80, about 90,about 95, about 100, about 120, about 130, about 140, about 150, about175, about 200, about 220, about 250, about 275, about 300, about 325,about 350, about 375, or about 400 amino acids. In some embodiments, theN-terminal truncation lacks no more than about 5, no more than about 10,no more than about 15, no more than about 20, no more than about 25, nomore than about 30, no more than about 35, no more than about 40, nomore than about 50, no more than about 60, no more than about 65, nomore than about 70, no more than about 75, no more than about 80, nomore than about 90, no more than about 95, no more than about 100, nomore than about 120, no more than about 130, no more than about 140, nomore than about 150, no more than about 175, no more than about 200, nomore than about 220, no more than about 250, no more than about 275, nomore than about 300, no more than about 325, no more than about 350, nomore than about 375, or no more than about 400 amino acids. In someembodiments, the C-terminal truncation lacks no more than about 5, nomore than about 10, no more than about 15, no more than about 20, nomore than about 25, no more than about 30, no more than about 35, nomore than about 40, no more than about 50, no more than about 60, nomore than about 65, no more than about 70, no more than about 75, nomore than about 80, no more than about 90, no more than about 95, nomore than about 100, no more than about 120, no more than about 130, nomore than about 140, no more than about 150, no more than about 175, nomore than about 200, no more than about 220, no more than about 250, nomore than about 275, no more than about 300, no more than about 325, nomore than about 350, no more than about 375, or no more than about 400amino acids. In some embodiments, the truncation comprises an N-terminaltruncation that removes at least about, at most about, or about 5, 10,15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, or 500 amino acids. In someembodiments, the truncation comprises a C-terminal truncation thatremoves at least about, at most about, or about 5, 10, 15, 20, 25, 30,40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, or 500 amino acids. In some embodiments, theN-terminal truncation, the C-terminal truncation, or both, may be morethan about 500 amino acids, more than about 1000 amino acids, more thanabout 1500 amino acids, more than about 2000 amino acids, more thanabout 5000 amino acids, more than about 10000 amino acids, more thanabout 100000 amino acids, more than about 1000000 amino acids.

In some embodiments, truncations of regions which do affect functionalactivity of a protein or enzyme may be engineered. In some embodiments,truncations of regions which do not affect functional activity of aprotein or enzyme may be engineered. A truncation may comprise atruncation of less than about 5, less than about 10, less than about 15,less than about 20, less than about 25, less than about 30, less thanabout 35, less than about 40, less than about 45, less than about 50,less than about 60, less than about 70, less than about 80, less thanabout 90, less than about 100, less than about 125, less than about 150,less than about 200, less than about 250, less than about 300, less thanabout 350, less than about 400 or more amino acids. A truncation maycomprise a truncation of more than about 5, more than about 10, morethan about 15, more than about 20, more than about 25, more than about30, more than about 35, more than about 40, more than about 45, morethan about 50, more than about 60, more than about 70, more than about80, more than about 90, more than about 100, more than about 125, morethan about 150, more than about 200, more than about 250, more thanabout 300, more than about 350, more than about 400 or more amino acids.A truncation may comprise a truncation of about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 87%, about 90%, about 92%, about 95% or about 100%of the polypeptide or enzyme.

In some embodiments, the variant or modified enzyme or modified proteinmay comprise one or more modification(s) at an amino acid position. Insome embodiments, a variant, a mutant, or modified polypeptides orenzymes of the present disclosure may possess an increased activity,such as an increased RNA-dependent DNA polymerase activity or aDNA-dependent DNA polymerase activity, compared to the correspondingunmutated or unmodified or wildtype polymerase or as compared to one ormore polymerases (e.g., RNA-dependent DNA polymerase, or a reversetranscriptase). In some embodiments, a polymerase or a reversetranscriptase having an increase in activity may be a modifiedpolymerase or a modified reverse transcriptase that has at least about a5% increase, at least about a 10% increase, at least about a 25%increase, at least about a 30% increase, at least about a 50% increase,at least about a 100% increase, at least about a 150% increase, at leastabout a 200% increase, at least about a 300% increase, at least about a500% increase, at least about a 1,000% increase, at least about a 2,500%increase or at least about a 5,000% increase as compared to (1) thecorresponding unmutated or wild-type enzyme; or (2) a particularpolymerase (e.g., RNA-dependent DNA polymerase, reverse transcriptase)or a particular reverse transcriptase, or a group of polymerases, or agroup of reverse transcriptases. In some embodiments, the modifiedpolymerase or the modified reverse transcriptase of the presentdisclosure may have an increase in activity of from about 5% to about5,000%, from about 5% to about 2,500%, from about 5% to about 1000%,from about 5% to about 500%, from about 5% to about 250%, from about 5%to about 100%, from about 5% to about 50%, from about 5% to about 25%,from about 25% to about 5,000%, from about 25% to about 2,500%, fromabout 25% to about 1,000%, from about 25% to about 500%, from about 25%to about 250%, from about 25% to about 100%, from about 100% to about5,000%, from about 100% to about 2,500%, from about 100% to about 1000%,from about 100% to about 500%, or from about 100% to about 250%. Anincrease in RNA-dependent DNA polymerase activity and/or DNA-dependentDNA polymerase for a modified polymerase or modified reversetranscriptase of the present disclosure may also be measured accordingto relative activity compared to (1) the corresponding unmodified orwild-type enzyme; or (2) a particular polymerase (e.g., RNA-dependentDNA polymerase, reverse transcriptase) or a particular reversetranscriptase, or a group of polymerases, or a group of reversetranscriptases. In some embodiments, the increase in such relativeactivity is at least about 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 75, 100,150, 200, 300, 500, 1,000, 2,500, 5,000, 10,000, or 25,000 fold when theactivity of a modified polymerase or modified reverse transcriptase ofthe present disclosure is compared to (1) the corresponding unmutated orwild-type enzyme; or (2) a particular polymerase (e.g., RNA-dependentDNA polymerase, reverse transcriptase) or a particular reversetranscriptase, or a group of polymerases, or a group of reversetranscriptases. Thus a modified polymerase or modified reversetranscriptase of the present disclosure may have an increasedRNA-dependent DNA polymerase and/or an increased DNA-dependent DNApolymerase activity of from about 1.1 fold to about 25,000 fold, fromabout 1.1 fold to about 10,000 fold, from about 1.1 fold to about 5,000fold, from about 1.1 fold to about 2,500 fold, from about 1.1 fold toabout 1,000 fold, from about 1.1 fold to about 500 fold, from about 1.1fold to about 250 fold, from about 1.1 fold to about 50 fold, from about1.1 fold to about 25 fold, from about 1.1 fold to about 10 fold, fromabout 1.1 fold to about 5 fold, from about 5 fold to about 25,000 fold,from about 5 fold to about 5,000 fold, from about 5 fold to about 1,000fold, from about 5 fold to about 500 fold, from about 5 fold to about100 fold, from about 5 fold to about 50 fold, from about 5 fold to about25 fold, from about 50 fold to about 25,000 fold, from about 50 fold toabout 5,000 fold, from about 50 fold to about 1,000 fold, from about 50fold to about 500 fold, from about 50 fold to about 100 fold, from about100 fold to about 25,000 fold, from about 1,000 fold to about 25,000fold, from about 4,000 fold to about 25,000 fold, from about 10,000 foldto about 25,000 fold, from about 15,000 fold to about 25,000 fold, fromabout 1,000 fold to about 10,000 fold, from about 2,500 fold, to about10,000 fold, from about 5,000 fold to about 10,000 fold, from about7,500 fold to about 10,000 fold, from about 1,000 fold to about 15,000fold, from about 2,500 fold, to about 15,000 fold, from about 5,000 foldto about 15,000 fold, from about 7,500 fold to about 15,000 fold, fromabout 10,000 fold to about 15,000 fold, or from about 12,500 fold toabout 15,000 fold.

In some embodiments, a modified enzyme, modified polypeptide havingreverse transcriptase activity, or a non-naturally occurring enzymeexhibits an altered (e.g., increased or decreased) processivity for agiven nucleotide substrate relative to an unmodified or naturallyoccurring counterpart. In some embodiments, the modified enzyme,modified polypeptide having reverse transcriptase activity, or anon-naturally occurring enzyme exhibits a processivity for a givennucleotide substrate that is at least about 5%, 10%, 25%, 37.5%, 50%,75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000% as high as the processivity of a reference enzyme for the samenucleotide substrate. In some embodiments, the reference enzyme is theunmodified counterpart of the modified enzyme. In some embodiments, thereference enzyme is a reverse transcriptase. In some embodiments, thereference enzyme is a non-LTR retrotransposon. In some embodiments, thereference enzyme is a LTR retrotransposon. In some embodiments, thereference enzyme is an R2 reverse transcriptase. In some embodiments,the reference enzyme or polypeptide comprises an amino acid sequence ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and/or SEQ IDNO: 48. In some embodiments, an R2 comprises at least one mutationand/or modification. In some embodiments, an R2 comprises an amino acidsequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, orSEQ ID NO: 67. In some embodiments, an R2 and/or a modified enzymeand/or a modified reverse transcriptase comprises a mutation at one ormore amino acids selected from (but not limited to) C952S, and/or C956S,and/or C952S, C956S (double mutant), and/or C969S, and/or H970Y, and/orR979Q, and/or R976Q, and/or R1071S, and/or R328A, and/or R329A, and/orQ336A, and/or R328A, R329A, Q336A (triple mutant), and/or G426A, and/orD428A, and/or G426A, D428A (double mutant), and/or any combinationthereof. In some embodiments, the amino acid position is based on SEQ IDNO: 52. In some embodiments, the amino acid position is based on awild-type reverse transcriptase. In some embodiments, the amino acidposition is based on a wild-type R2. In some embodiments, a cysteine ismutated. In some embodiments, a cysteine is mutated to a serine. In someembodiments, an arginine is mutated. In some embodiments, an arginine ismutated to a serine, or a glutamine, or an alanine. In some embodiments,an amino acid is mutated to an alanine. In some embodiments, a glutamineis mutated to an alanine. In some embodiments, an aspartic acid ismutated to an alanine. In some embodiments, a glycine is mutated to analanine. In some embodiments, a histidine is mutated to a tyrosine.

In some embodiments, a variant, or a modified enzyme (e.g., modifiedreverse transcriptase), or a modified polypeptide having reversetranscriptase activity of the present disclosure comprises at least onealtered characteristic that improves enzyme property. In someembodiments, a variant or a modified enzyme (e.g., modified reversetranscriptase) or a modified polypeptide having reverse transcriptaseactivity of the present disclosure comprises at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, atleast about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, and at least about 99%sequence identity to an amino acid sequence disclosed in the presentdisclosure. In some embodiments, a variant, or a modified enzyme, or amodified polypeptide having reverse transcriptase activity comprises atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, and/orat least about 99% sequence identity to an amino acid sequencecorresponding to a GenBank number in TABLE 1. In some embodiments, avariant, or a modified enzyme, or a modified polypeptide having reversetranscriptase activity comprises at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 81%, at least about 82%, at leastabout 83%, at least about 84%, at least about 85%, at least about 86%,at least about 87%, at least about 88%, at least about 89%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, and/or at least about 99%, sequenceidentity to an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8 SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ IDNO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQID NO: 46, SEQ ID NO: 47, and/or SEQ ID NO: 48. In some embodiments, avariant, or a modified enzyme, or a modified polypeptide having reversetranscriptase activity comprises at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 81%, at least about 82%, at leastabout 83%, at least about 84%, at least about 85%, at least about 86%,at least about 87%, at least about 88%, at least about 89%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, and/or at least about 99% sequenceidentity to an amino acid sequence set forth in SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO: 66, and/or SEQ ID NO: 67. See TABLE 2. SEQID NO: 49 is an example of an R2 RT C-truncation, SEQ ID NO: 50 is anexample of an R2 RT N-truncation, SEQ ID NO: 51 is an example of an R2RT N- and C-truncation, SEQ ID NO: 52 is an example of an R2 wild type.SEQ ID NO: 53-67 are examples of R2 with single, double, or triplemutations (TABLE 2). In some embodiments, the modified reversetranscriptase is derived from an arthropod. In some embodiments, thearthropod is Bombyx mori.

TABLE 1 GenBank Description GI number SEQ ID NO: AAB59214.1 reversetranscriptase-like protein [Bombyx 903695 1 mori] T18197 reversetranscriptase-like protein - 7511784 2 silkworm KMQ90176.1 reversetranscriptase [Lasius niger] 861630869 3 KYB24671.1 Retrovirus-relatedPol polyprotein from 1004394526 4 type-2 retrotransposable element R2DM-like Protein [Tribolium castaneum] KMQ90064.1 reverse transcriptase[Lasius niger] 861630480 5 ACJ71597.1 reverse transcriptase[Rhynchosciara 215982090 6 americana] AFM44926.1 R2 protein[Eyprepocnemis plorans] 391862173 7 AHN53448.1 reverse transcriptase[Nuttalliella 599127491 8 namaqua] ACJ46647.1 reverse transcriptase[Triops cancriformis] 213399743 9 BAC82590.1 reverse transcriptase[Ciona intestinalis] 34392525 10 AAB94032.1 reverse transcriptase domainprotein 2735957 11 [Drosophila mercatorum] AAC34906.1 reversetranscriptase [Forficula auricularia] 3559776 12 BAC82589.1 reversetranscriptase [Ciona intestinalis] 34392523 13 AIL01110.1 reversetranscriptase [Bacillus rossius] 674275091 14 AFO19998.1 R2 protein[Lepidurus couesii] 397174834 15 KMQ88340.1 reverse transcriptase[Lasius niger] 861624704 16 AFO19997.1 R2 protein [Lepidurus couesii]397174832 17 AAC34903.1 reverse transcriptase [Anurida maritima] 355977018 AFO19995.1 R2 protein [Lepidurus apus lubbocki] 397174828 19BAC82591.1 reverse transcriptase [Ciona intestinalis] 34392527 20CAX83712.1 endonuclease-reverse transcriptase 254587310 21 [Schistosomajaponicum] XP_009165216.1 hypothetical protein T265_13057 684375893 22[Opisthorchis viverrini] AAB94040.1 reverse transcriptase [Hippodamia2736050 23 convergens] AAV85443.1 reverse transcriptase-like protein56267941 24 [Amblyomma americanum] AAV85445.1 reverse transcriptase-likeprotein [Ixodes 56267945 25 scapularis] KRY44798.1 Retrovirus-relatedPol polyprotein from 954380717 26 type-2 retrotransposable element R2DM[Trichinella britovi] AAV85444.1 reverse transcriptase-like protein56267943 27 [Rhipicephalus microplus] KRX52183.1 Retrovirus-related Polpolyprotein from 954258524 28 type-2 retrotransposable element R2DM[Trichinella sp. T9] KRX72028.1 Retrovirus-related Pol polyprotein from954280597 29 type-2 retrotransposable element R2DM [Trichinella sp. T6]AFO19999.1 R2 protein [Lepidurus couesii] 397174836 30 AAA21258.1reverse transcriptase [Drosophila ambigua] 533135 31 KRX12851.1Retrovirus-related Pol polyprotein from 954202918 32 type-2retrotransposable element R2DM [Trichinella nelsoni] KFD59471.1hypothetical protein M514_11684 669318869 33 [Trichuris suis] KXZ75771.1hypothetical protein TcasGA2_TC031700 1004173031 34 [Triboliumcastaneum] XP_002412745.1 reverse transcriptase, putative [Ixodes241683764 35 scapularis] BAE46603.1 reverse transcriptase [Eptatretusburgeri] 77799487 36 AFO20000.1 R2 protein [Triops cancriformis]397174838 37 KRX36111.1 Retrovirus-related Pol polyprotein from954240338 38 type-2 retrotransposable element R2DM [Trichinellamurrelli] KRZ66264.1 Retrovirus-related Pol polyprotein from 95458856739 type-2 retrotransposable element R2DM [Trichinella papuae] KRY45664.1Retrovirus-related Pol polyprotein from 954382014 40 type-2retrotransposable element R2DM [Trichinella britovi] CAJ00246.1 TPA:polyprotein [Schistosoma mansoni] 67625717 41 Q03278 Retrovirus-relatedPol polyprotein from 2851550 42 type-1retrotransposable element R2[Nasonia vitripennis (Parasitic wasp)] Q03279 Retrovirus-related Polpolyprotein from 548546 43 type-1 retrotransposable element R2 [Bradysiacoprophila (Dark-winged fungus gnat) (Sciara coprophila)] KXZ75830.1hypothetical protein TcasGA2_TC031908 1004173289 44 [Triboliumcastaneum] P16423.1 RecName: Full = Retrovirus-related Pol 130551 45polyprotein from type-2 retrotransposable element R2DM [Drosophilamelanogaster] KRX34481.1 Retrovirus-related Pol polyprotein from954238165 46 type-2 retrotransposable element R2DM [Trichinellamurrelli] EEB15300.1 [Pediculus humanus corporis] - reverse 212512557 47transcriptase, putative XP_002431867.1 reverse transcriptase, putative[Pediculus 48 humanus corporis]

TABLE 2 SEQ ID Description Sequence NO: Example of a C-MAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPD 49 terminalGCTRGKHVTAAPMDGPRGPSSLAGTFGWGLAIPAG truncationEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESE RTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGE EIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEP DFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGP DGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQGGGVG Example of N-MAHHHHHHVGTVGTGGGSGGASTALKTAGRRNDL 50 terminalHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAA truncationEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFD WRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRFNQMTSVMGGGVG Example of N-,MAHHHHHHVGTVGTGGGSGGASTALKTAGRRNDL 51 C-truncationHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFD WRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTR TPTSTKWIRERCAQGGGVG R2 wild typeMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPD 52GCTRGKHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNR GLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVP ILALRGSHMNWTRFNQMTSVMGGGVGC952S MAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 53KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTSRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* C956SMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 54KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGSKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* C952S, C956SMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 55KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTSRAGSKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* C969SMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 56KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQSHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* H970YMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 57KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCYRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* R979QMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 58KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILQHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* R976QMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 59KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGQILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* R1071SMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 60KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVSATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* R328AMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 61KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKARAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* R329AMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 62KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRAAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* Q336AMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 63KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVAELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* R328A, R329A,MAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 64 Q336AKHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKAAAEYARVAELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* G426AMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 65KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPAPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* D428AMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 66KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPAGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG* G426A, D428AMAHHHHHHVGTVGTGGGSGGASTALSLMGRCNPDGCTRG 67KHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPAPAGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRF NQMTSVMGGGVG*

In some embodiments, the polypeptides, proteins, enzymes, modifiedenzymes (e.g., modified reverse transcriptase), modified polypeptides,non-naturally occurring enzymes, or variants comprise a fusion with, butnot limited to, a protein, a domain, a fusion partner, a carrierprotein, a target sequence, an antigenic determinant, or any combinationthereof. In some embodiments, the reverse transcriptase or modifiedreverse transcriptase is fused to a protein, a domain, a fusion partner,a target sequence, an antigenic determinant, or any combination thereof.In some embodiments, the non-LTR retrotransposon or modified non-LTRretrotransposon is fused to a protein, a domain, a fusion partner, atarget sequence, an antigenic determinant, or any combination thereof.In some embodiments, the modified LTR retrotransposon is fused to aprotein, a domain, a fusion partner, a target sequence, an antigenicdeterminant, or any combination thereof. In some embodiments, themodified R2 non-LTR retrotransposon is fused to a protein, a domain, afusion partner, a target sequence, an antigenic determinant, or anycombination thereof. In some embodiments, the modified R2 reversetranscriptase is fused to a protein, a domain, a fusion partner, atarget sequence, an antigenic determinant, or any combination thereof.In some embodiments, the modified reverse transcriptase is fused to aprotein, a domain, a fusion partner, a target sequence, an antigenicdeterminant, or any combination thereof. In some embodiments, thevariant is fused to a protein, a domain, a fusion partner, a targetsequence, an antigenic determinant, or any combination thereof. In someembodiments, the polypeptide having reverse transcriptase activity isfused to a protein, a domain, a fusion partner, a target sequence, anantigenic determinant, or any combination thereof.

In some embodiments, the fused polypeptides, proteins, enzymes, modifiedenzymes (e.g., modified reverse transcriptase), modified polypeptides,non-naturally occurring enzymes, or variants thereof increase stability(e.g., increase thermostability), increase shelf life, increase activefraction(s), and/or improve purification compared to the wild-typecounterpart, naturally occurring enzyme, or unfused polypeptides,proteins, enzymes, or variants thereof. In some embodiments, a modifiedreverse transcriptase comprises a fusion partner or a carrier protein.In some embodiments, the selection of the fusion protein, domain, fusionpartner, target sequence, antigenic determinant, or any combinationthereof is based on the mechanism causing reduced or increased stability(e.g., increased thermostability), reduced or increased shelf life,and/or reduced or increased expression level (Costa et al., “Fusion tagsfor protein solubility, purification and immunogenicity in Escherichiacoli: the novel Fh8 system. Front Microbiol. 2014 Feb. 19; 5:63). Insome embodiments, the fusion tags enhance the solubility of theirpartner proteins. In some embodiments, the fusion proteins formmicelle-like structures. In some embodiments, the micelle-likestructures are misfolded or unfolded proteins that are sequestered andprotected from the solvent and/or the soluble protein domains faceoutward. In some embodiments, the fusion partners attract chaperones. Insome embodiments, the fusion tag drives its partner protein into achaperone-mediated folding pathway. In some embodiments, the MBP and/orN-utilization substance (NusA) are two fusion tags that present thismechanism. In some embodiments, the fusion partners have an intrinsicchaperone-like activity. In some embodiments, the hydrophobic patches ofthe fusion tag interact with partially folded passenger proteins,preventing self-aggregation, and promoting proper folding. In someembodiments, the solubility enhancer partners may play a passive role inthe folding of their target proteins, reducing the chances for proteinaggregation. In some embodiments, the fusion partners net charges. Insome embodiments, the highly acidic fusion partners inhibit proteinaggregation. In some embodiments, the fusion is with, but it is notlimited to, Fh8, MBP, NusA, Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT,Skp, T7PK, EspA, Mocr, Ecotin, CaBP, ArsC, IF2-domain I, an expressivitytag, an expressivity tag that is part of IF2-domain I, RpoA, SlyD, Tsf,RpoS, PotD, Crr, msyB, yjgD, rpoD, His6, or any combination thereof. Insome embodiments, the fusion enhances protein solubility and/orpurification. In some embodiments, the Fh8 may act as an effectivesolubility enhancer partner and/or robust purification. In someembodiments, the Fh8 fusion tag has an amino acid sequence comprisingMPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSI LSS.In some embodiments, the codon optimized sequence comprisesATGCCGTCTGTTCAGGAAGTTGAAAAACTGCTGCACGTTCTGGACCGTAACGGTGACGGTAAAGTTTCTGCGGAAGAACTGAAAGCGTTCGCGGACGACTCTAAATGCCCGCTGGACTCTAACAAAATCAAAGCGTTCATCAAAGAACACGACAAAAACAAAGACGGTAAACTGGACCTGAAAGAACTGGTTTCTATCCTGTCTTCTTAG. In some embodiments, an enzyme, or a modifiedenzyme (e.g., modified reverse transcriptase), or a protein (e.g.,modified protein), or a polypeptide (e.g., modified polypeptide), or avariant, or a product, or a nucleic acid molecule, or a cDNA molecule,or a template, or an acceptor nucleic acid molecule, or a primer, or anRNA, or a DNA, or a fragment nucleic acid, or a degraded nucleic acid,of the present disclosure may comprise one or more tag(s). In someembodiments, the fragmented or degraded RNA or DNA, or a variant thereofmay comprise one or more tag(s). In some embodiments, the R2 reversetranscriptase, or a variant thereof, may comprise one or more tag(s). Insome embodiments, the non-LTR retrotransposon protein or polypeptidehaving reverse transcriptase activity, or a variant thereof, maycomprise one or more tag(s). In some embodiments, the cDNA molecule maycomprise one or more tag(s). In some embodiments, the tag may becaptured on a solid support, facilitating the isolation of the enzyme,or protein, or polypeptide, or a variant, or a product of the presentdisclosure. In some embodiments, the tag may be biotin that can berecognized by avidin. The affinity tag may include multiple biotinresidues for increased binding to multiple avidin molecules. In someembodiments, the tag may include a functional group such as an azidogroup or an acetylene group, which enables capture through copper(I)mediated click chemistry (see H. C. Kolb and K. B. Sharpless, DrugDiscovery Today, 2003, 8(24), 1128-1137). In some embodiments, the tagmay include an antigen that may be captured by an antibody bound on asolid support. In some embodiments, the tag may include, but is notlimited to, His-tag, His6-tag, Calmodulin-tag, CBP, CYD (covalent yetdissociable NorpD peptide), Strep II, FLAG-tag, HA-tag, Myc-tag, S-tag,SBP-tag, Softag-1, Softag-3, V5-tag, Xpress-tag, Isopeptag, SpyTag, B,HPC (heavy chain of protein C) peptide tags, GST, MBP, biotin, biotincarboxyl carrier protein, glutathione-S-transferase-tag, greenfluorescent protein-tag, maltose binding protein-tag, Nus-tag,Strep-tag, thioredoxin-tag, and combinations thereof. In someembodiments, the tagged molecule may be subjected to sequencing.

In some embodiments, a molecular barcode may be attached to any regionof a molecule. For example, the molecular barcode may be attached to the5′ or 3′ end of a polynucleotide (e.g., DNA, RNA). For example, thetarget-specific region of the molecular barcode comprises a sequencethat is complementary to a sequence in the 5′ region of the molecule.The target-specific region of the molecular barcode may also comprise asequence that is complementary to a sequence in the 3′ region of themolecule. In some instances, the molecular barcode is attached a regionwithin a gene or gene product. For example, genomic DNA is fragmentedand a sample tag or molecular identifier label is attached to thefragmented DNA. In other instances, an RNA molecule is alternativelyspliced and the molecular barcode is attached to the alternativelyspliced variants. In another example, the polynucleotide is digested andthe molecular barcode is attached to the digested polynucleotide. Inanother example, the target-specific region of the molecular barcodecomprises a sequence that is complementary to a sequence within themolecule.

In some embodiments the method of the present disclosure comprisesintroducing a biotin moiety or another affinity purification moiety to,for example, a nucleic acid molecule, such as DNA, RNA, or a combinationof DNA and RNA. In some embodiments, the method further comprisesimmobilizing the affinity purification tagged nucleic acid molecule on asolid support. In some embodiments the solid support is a sepharoseresin or magnetic beads having an affinity purification material, suchas avidin, streptavidin, chitin, glutathione and the like, boundthereto.

In some embodiments, the enzyme, or protein, or polypeptide, or avariant, or a product of the present disclosure may be bound to a solidsupport. In some embodiments, the fragmented or degraded nucleic acid(e.g., RNA or DNA), or a variant thereof may be bound to a solidsupport. In some embodiments, the R2 reverse transcriptase, or a variantthereof, may be bound to a solid support. In some embodiments, thenon-LTR retrotransposon protein or polypeptide having reversetranscriptase activity, or a variant thereof, may be bound to a solidsupport. In some embodiments, the cDNA molecule may be bound to a solidsupport. In some embodiments, the solid support may be glass, plastic,porcelain, resin, sepharose, silica, or other material. In someembodiments, the solid support may be a plate that is substantially flatsubstrates, gel, microbeads, magnetic beads, membrane, or other suitableshape and size. In some embodiments, the microbeads may have diameterbetween 10 nm to several millimeters. In some embodiments, the solidsupport may be non-porous or porous with various density and size ofpores. In some embodiments the DNA and/or RNA fragment may be capturedon a solid support, unwanted DNA and/or RNA may be washed away. In someembodiments, the DNA and/or RNA fragment may be released from the solidsupport, for example, by using restriction enzyme.

In some embodiments, the solid support may comprise the target nucleicacid binding region, wherein the target nucleic acid binding regioncomprises a sequence selected from the group consisting of agene-specific sequence, an oligo-dT sequence, a random multimer, and anycombination thereof. In some embodiments, the solid support furthercomprises a target nucleic acid or complement thereof. In someembodiments, the solid support comprises a plurality of target nucleicacids or complements thereof comprising from about 0.01% to about 100%of transcripts of a transcriptome of an organism or complements thereof,or from about 0.01% to about 100% of genes of a genome of an organism orcomplements thereof. In some embodiments, the cellular labels of theplurality of oligonucleotides comprise a first random sequence connectedto a second random sequence by a first label linking sequence; and themolecular labels of the plurality of oligonucleotides comprise randomsequences. In some embodiments, the solid support is selected from thegroup consisting of a polydimethylsiloxane (PDMS) solid support, apolystyrene solid support, a glass solid support, a polypropylene solidsupport, an agarose solid support, a gelatin solid support, a magneticsolid support, a pluronic solid support, and any combination thereof. Insome embodiments, the plurality of oligonucleotides comprise a linkercomprising a linker functional group, and the solid support comprises asolid support functional group; wherein the solid support functionalgroup and linker functional group connect to each other. In someembodiments, the linker functional group and the solid supportfunctional group are individually selected from the group consisting ofC6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), andany combination thereof. In some embodiments, molecular labels of theplurality of oligonucleotides comprise at least 15 nucleotides.

In some embodiments, fusion partners may be removed from their targetprotein by enzymatic cleavage, chemical cleavage, and/or by using an invivo cleavage strategy. In some embodiments, proteases may be used fortag removal. In some embodiments, the protease may be an endoprotease,serine protease, factor Xa, enterokinase, alpha-thrombin, a viralprotease, tobacco etch virus (TEV), the human rhinovirus 3C protease,SUMO protease, exoprotease, metallocarboxypeptidase, or aminopeptidase.In some embodiments, a fusion tag may be removed by two purificationsteps. In some embodiments, the initial affinity purification stepincludes (e.g., via a histidine tag located at the N-terminal of thefusion protein), the purified fusion protein mixed in solution with theendoprotease (e.g., a his-tagged protease) to cleave off the tag. Thecleaved target protein may be recovered in the flow-through sample aftera second affinity purification step, in which the cleaved fusion tag andthe added protease are collected in the eluted sample.

In some embodiments, the modified enzyme, modified reversetranscriptase, non-naturally occurring enzyme, or modified polypeptidehaving reverse transcriptase activity of the present disclosure showsactivity, is capable of template jumping, and/or generate a nucleic acidmolecule (e.g., cDNA molecule) without thermal cycling. In someembodiments, the modified reverse transcriptase, modified enzyme,non-naturally occurring enzyme, or the modified polypeptide havingreverse transcriptase activity of the present disclosure shows activity,is capable of template jumping, and/or generate a nucleic acid, cDNAmolecule at a temperature ranging from about 25° C. to about 42° C.,from about 12° C. to about 42° C., from about 8° C. to about 50° C.,from about 4° C. to about 60° C., from about 27° C. to about 35° C.,from about 28° C. to about 33° C., from about 29° C. to about 32° C.,from about 30° C. to about 37° C., from about 26° C. to about 38° C.,from about 30° C. to about 37° C., from about 25° C. to about 32° C.,from about 29° C. to about 31° C., from about 27° C. to about 38° C.,from about 29° C. to about 38° C. In some embodiments, the non-naturallyoccurring enzyme, modified reverse transcriptase, modified enzyme, ormodified polypeptide having reverse transcriptase activity of thepresent disclosure shows activity, is capable of template jumping,and/or generate a nucleic acid molecule (e.g., cDNA molecule) at about30° C., or at about 35° C., or at about 25° C. In some embodiments, themodified enzyme, modified reverse transcriptase, non-naturally occurringenzyme, or modified polypeptide having reverse transcriptase activity ofthe present disclosure shows activity, is capable of template jumping,and/or generate a nucleic acid molecule (e.g., cDNA molecule) at atemperature equal to less than about 38° C., equal to less than about42° C., equal to less than about 50° C., equal to less than about 60°C., equal to less than about 35° C., equal to less than about 30° C.,equal to less than about 28° C., equal to less than about 25° C., equalto less than about 20° C., equal to less than about 12° C., equal toless than about 8° C., or equal to less than about 4° C. In someembodiments, the modified enzyme, modified reverse transcriptase,non-naturally occurring enzyme, or modified polypeptide having reversetranscriptase activity of the present disclosure shows activity, iscapable of template jumping, and/or generate a nucleic acid molecule(e.g., cDNA molecule) at a temperature equal to less than about 36° C.In some embodiments, the modified enzyme, modified reversetranscriptase, non-naturally occurring enzyme, or modified polypeptidehaving reverse transcriptase activity of the present disclosure showsactivity, is capable of template jumping, and/or generate a nucleic acidmolecule (e.g., cDNA molecule) at room temperature. In some embodiments,the modified enzyme, modified reverse transcriptase, non-naturallyoccurring enzyme, or modified polypeptide having reverse transcriptaseactivity of the present disclosure shows activity, is capable oftemplate jumping, and/or generate a nucleic acid molecule (e.g., cDNAmolecule) at a temperature of at about or of at most about 8° C., atabout or of at most about 12° C., at about or of at most about 20° C.,at about or of at most about 25° C., at about or of at most about 28°C., at about or of at most about 30° C., at about or of at most about31° C., at about or of at most about 32° C., at about or of at mostabout 33° C., at about or of at most about 34° C., at about or of atmost about 35° C., at about or of at most about 36° C. at about or of atmost about 39° C., at about or of at most about 40° C., at about or ofat most about 41° C., at about or of at most about 42° C., at about orof at most about 50° C., at about or of at most about 55° C., at aboutor of at most about 60° C. In some embodiments, the modified enzyme,modified reverse transcriptase, non-naturally occurring enzyme, ormodified polypeptide having reverse transcriptase activity of thepresent disclosure shows activity, is capable of template jumping,and/or generate a nucleic acid molecule (e.g., cDNA molecule) at atemperature equal to or less than about any temperature between about42° C. to about 80° C., or between about 35° C. to about 80° C., orbetween about 30° C. to about 50° C., or between about 8° C. to about50° C., or between about 12° C. to about 42° C.

In some embodiments, a modified enzyme, modified reverse transcriptase,modified polypeptide having reverse transcriptase activity, or anon-naturally occurring enzyme of the present disclosure has at leastone altered characteristic relative to an unmodified or naturallyoccurring enzyme. In some embodiments, the altered characteristicenables the modified enzyme, modified reverse transcriptase,non-naturally occurring enzyme, or modified polypeptide having reversetranscriptase activity to generate a nucleic acid molecule and/or acomplementary deoxyribonucleic acid (cDNA) molecule from a templatenucleic acid molecule without thermal cycling. In some embodiments, amodified enzyme, modified reverse transcriptase, modified polypeptidehaving reverse transcriptase activity, or a non-naturally occurringenzyme of the present disclosure is capable of generating one or morecopies of the nucleic acid molecule or cDNA molecule at an error rate ofat most about 0.5%, of at most about 1%, of at most about 1.5%, of atmost about 2%, of at most about 2.5%, of at most about 3%, of at mostabout 3.5%, of at most about 4%, of at most about 4.5%, of at most about5%, of at most about 6%, of at most about 7%, of at most about 8%, of atmost about 9%, of at most about 10%, of at most about 15%, of at mostabout 20%, of at most about 25%, of at most about 30%, of at most about40%, of at most about 45%, of at most about 50%, of at most about 60%,of at most about 65%, of at most about 70%, of at most about 75%, or ofat most about 80%. In some embodiments, the modified enzyme, modifiedreverse transcriptase, modified polypeptide having reverse transcriptaseactivity, or the non-naturally occurring enzyme of the presentdisclosure is a variant of any one of the sequences disclosed herein. Insome embodiments, the modified enzyme, modified reverse transcriptase,modified polypeptide having reverse transcriptase activity, or thenon-naturally occurring enzyme of the present disclosure is a variant ofany one of the sequences corresponding to accession numbers provided inTABLE 1. In some embodiments, the modified enzyme, modified reversetranscriptase, modified polypeptide having reverse transcriptaseactivity, or the non-naturally occurring enzyme of the presentdisclosure is a variant of any one of the sequences provided in SEQ IDNos: 1-67. In some embodiments, a modified enzyme, modified reversetranscriptase, modified polypeptide having reverse transcriptaseactivity, or a non-naturally occurring enzyme of the present disclosurehas at least one altered characteristic that improves enzyme propertyrelative to an unmodified or a naturally occurring enzyme. In someembodiments, the at least one altered characteristic that improvesenzyme property comprises at least one of increased/improved stability(e.g., increased/improved thermostability), increased/improved specificactivity, increased/improved protein expression, increased/improvedpurification, increased/improved processivity, increased/improved stranddisplacement, increased/improved template jumping, andincreased/improved fidelity.

In one embodiment, the present disclosure relates to a non-naturallyoccurring enzyme that subjects a template nucleic acid molecule toreverse transcription to generate a complementary deoxyribonucleic acid(cDNA) product and amplification of the cDNA product at a processivityof at least about 80%, at least about 85%, at least about 87%, at leastabout 90%, at least about 92%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or at leastabout 99.5% per base as measured at about 12° C., about 15° C., about20° C., about 25° C., about 30° C., about 32° C., about 35° C., about40° C.

In some embodiments, the non-naturally occurring enzyme has aperformance index greater than about 1.0 for at least one enzymeproperty. In some embodiments, enzyme property is at least one of thegroup consisting of improved stability (e.g., improved thermostability),specific activity, protein expression, purification, processivity,strand displacement, template jumping, increased DNA/RNA affinity, andfidelity.

In one embodiment, the present disclosure relates to a non-naturallyoccurring enzyme that subjects a template nucleic acid molecule toreverse transcription to generate a complementary deoxyribonucleic acid(cDNA) product, a nucleic acid product, and amplification of the cDNAproduct in a time period of about 3 hours or less and/or at aperformance index greater than about 1.0 for at least one enzymeproperty selected from the group consisting of improved stability (e.g.,improved thermostability), specific activity, protein expression,purification, processivity, strand displacement, template jumping,increased DNA/RNA affinity, and fidelity. In some embodiments, thetemperature is from about 25° C. to about 40° C. (e.g., about 28° C.,about 30° C., about 32° C., about 35° C., or about 37° C.). In someembodiments, the temperature is from about 8° C. to about 50° C. (e.g.,about 8° C., about 20° C., about 42° C., about 45° C., or about 50° C.).

In one embodiment, the present disclosure relates to a non-naturallyoccurring enzyme that subjects a template nucleic acid molecule toreverse transcription to generate a complementary deoxyribonucleic acid(cDNA) product and amplification of the cDNA product in a time period of3 hours or less (e.g., 2.5 hours or less, 2 hours or less, 1.5 hours orless, 1 hour or less, or 30 minutes or less) and/or at a processivityfor a given nucleotide substrate that is at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 85%, at least about 88%, at least about 90%,at least about 95%, or at least about 98% higher than the processivityof a reference enzyme for the same nucleotide substrate.

In one embodiment, the present disclosure relates to a non-naturallyoccurring enzyme that subjects a template nucleic acid molecule toreverse transcription to generate a nucleic acid product andamplification of the nucleic acid product in a time period of 3 hours orless (e.g., 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1hour or less, or 30 minutes or less) and/or at a processivity for agiven nucleotide substrate that is at least about 5%, at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 85%, at least about 88%, at least about 90%, atleast about 95%, or at least about 98% higher than the processivity of areference enzyme for the same nucleotide substrate.

In one embodiment, the present disclosure provides a method ofamplifying a nucleic acid molecule, comprising subjecting the nucleicacid molecule to nucleic acid amplification using a modified reversetranscriptase. In some embodiments, the reverse transcriptase is capableof amplifying the nucleic acid molecule at processivity of at leastabout 80%, at least about 88%, at least about 90%, at least about 95%,or at least about 98% per base at about 4° C., about 8° C., about 12°C., about 30° C., about 28° C., about 29° C., about 32° C., about 35°C., about 37° C., about 42° C., about 50° C., or higher than about 42°C.

In some embodiments, the method of the present disclosure furthercomprises using the modified reverse transcriptase to subject a templatenucleic acid molecule to reverse transcription to yield the nucleic acidmolecule. In some embodiments, the nucleic acid molecule is a cell-freenucleic acid molecule. In some embodiments, the template nucleic acidmolecule is a cell-free nucleic acid molecule.

In some embodiments, primer extension or elongation reactions areutilized to generate amplified product. Primer extension/elongationreactions may comprise a cycle of incubating a reaction mixture at adenaturation temperature for a denaturation duration and incubating areaction mixture at an elongation temperature for an elongationduration.

Any type of nucleic acid amplification reaction may be used to amplify atarget nucleic acid and generate an amplified product. Moreover,amplification of a nucleic acid may linear, exponential, or acombination thereof. Amplification may be emulsion based or may benon-emulsion based. Non-limiting examples of nucleic acid amplificationmethods include reverse transcription, primer extension, polymerasechain reaction, ligase chain reaction, helicase-dependent amplification,asymmetric amplification, rolling circle amplification, and multipledisplacement amplification (MDA). In some embodiments, the amplifiedproduct may be DNA. In cases where a target RNA is amplified, DNA can beobtained by reverse transcription of the RNA and subsequentamplification of the DNA can be used to generate an amplified DNAproduct. The amplified DNA product may be indicative of the presence ofthe target RNA in the biological sample. In cases where DNA isamplified, any DNA amplification may be employed. Non-limiting examplesof DNA amplification methods include polymerase chain reaction (PCR),variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR,asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR,helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR,methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR,overlap-extension PCR, thermal asymmetric interlaced PCR, touchdownPCR), and ligase chain reaction (LCR). In some cases, DNA amplificationis linear. In some cases, DNA amplification is exponential. In somecases, DNA amplification is achieved with nested PCR, which can improvesensitivity of detecting amplified DNA products.

Denaturation temperatures may vary depending upon, for example, theparticular biological sample analyzed, the particular source of targetnucleic acid (e.g., viral particle, bacteria) in the biological sample,the reagents used, and/or the desired reaction conditions. In someembodiments, a denaturation temperature may be from about 80° C. toabout 110° C. In some embodiments, a denaturation temperature may befrom about 90° C. to about 100° C. In some embodiments, a denaturationtemperature may be from about 90° C. to about 97° C. In some examples, adenaturation temperature may be from about 92° C. to about 95° C. Instill other examples, a denaturation temperature may be about 80°, 81°C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90°C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99°C., or 100° C.

Denaturation durations may vary depending upon, for example, theparticular biological sample analyzed, the particular source of targetnucleic acid (e.g., viral particle, bacteria) in the biological sample,the reagents used, and/or the desired reaction conditions. In someembodiments, a denaturation duration may be less than or equal to about300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2seconds, or 1 second. For example, a denaturation duration may be nomore than about 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1second.

Elongation or extension temperatures may vary depending upon, forexample, the particular biological sample analyzed, the particularsource of target nucleic acid (e.g., viral particle, bacteria) in thebiological sample, the reagents used, and/or the desired reactionconditions. In some embodiments, an elongation temperature may be fromabout 30° C. to about 80° C. In some embodiments, an elongationtemperature may be from about 35° C. to about 72° C. In someembodiments, an elongation temperature may be from about 45° C. to about68° C. In some embodiments, an elongation temperature may be from about35° C. to about 65° C. In some embodiments, an elongation temperaturemay be from about 40° C. to about 67° C. In some embodiments, anelongation temperature may be from about 50° C. to about 68° C. In someembodiments, an elongation temperature may be about 0° C., 1° C., 2° C.,3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C.,13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 34° C., 33° C.,32° C., 31° C., 30° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C.,26° C., 27° C., 28° C., 29° C., 35° C., 36° C., 37° C., 38° C., 39° C.,40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C.,49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C.,58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C.,67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C.,76° C., 77° C., 78° C., 79° C., or 80° C.

Elongation durations may vary depending upon, for example, theparticular biological sample analyzed, the particular source of targetnucleic acid (e.g., viral particle, bacteria) in the biological sample,the reagents used, and/or the desired reaction conditions. In someembodiments, an elongation duration may be less than or equal to about360 seconds, less than or equal to about 300 seconds, 240 seconds, 180seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. In someembodiments, an elongation duration may be no more than about 120seconds, 90 seconds, 80 seconds, 70 seconds, 65 seconds, 60 seconds, 55seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1second.

In some embodiments, multiple cycles of a primer extension reaction canbe conducted. Any suitable number of cycles may be conducted. In someembodiments, the number of cycles conducted may be less than about 100,90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 cycles. The number of cyclesconducted may depend upon, for example, the number of cycles (e.g.,cycle threshold value (Ct)) necessary to obtain a detectable amplifiedproduct (e.g., a detectable amount of amplified DNA product that isindicative of the presence of a target RNA in a biological sample). Insome embodiments, the number of cycles necessary to obtain a detectableamplified product (e.g., a detectable amount of DNA product that isindicative of the presence of a target RNA in a biological sample) maybe less than about or about 100 cycles, 75 cycles, 70 cycles, 65 cycles,60 cycles, 55 cycles, 50 cycles, 40 cycles, 35 cycles, 30 cycles, 25cycles, 20 cycles, 15 cycles, 10 cycles, 8 cycles, 7 cycles, 5 cycles,or 4 cycles. Moreover, in some embodiments, a detectable amount of anamplifiable product (e.g., a detectable amount of DNA product that isindicative of the presence of a target RNA in a biological sample) maybe obtained at a cycle threshold value (Ct) of less than 100, 75, 70,65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2,1.

In some embodiments, an amplification step (e.g., primer amplification,template amplification, nucleic acid amplification) comprises a PCRstep. In some embodiments, each PCR cycle may comprise a denaturingstep, an annealing step, and an extension step. In some embodiments,each PCR cycle may comprise a denaturing step and an extension step. Insome embodiments, the PCR comprises at least about or about or at mostabout 1 cycle, at least about or about or at most about 4 cycles, atleast about or about or at most about 5 cycles, at least about or aboutor at most about 10 cycles, at least about or about or at most about 15cycles, at least about or about or at most about 20 cycles, at leastabout or about or at most about 25 cycles, at least about or about or atmost about 30 cycles, at least about or about or at most about 35cycles, at least about or about or at most about 40 cycles, at leastabout or about or at most about 45 cycles, at least about or about or atmost about 50 cycles, at least about or about or at most about 55cycles, at least about or about or at most about 60 cycles, at leastabout or about or at most about 65 cycles, at least about or about or atmost about 70 cycles, at least about or about or at most about 75cycles, at least about or about or at most about 80 cycles, at leastabout or about or at most about 90 cycles, at least about or about or atmost about 95 cycles, at least about or about or at most about 100cycles, at least about or about or at most about 110 cycles, at leastabout or about or at most about 120 cycles, at least about or about orat most about 130 cycles, at least about or about or at most about 140cycles, at least about or about or at most about 150 cycles, at leastabout or about or at most about 160. In some embodiments, the PCRcomprises from about 10 cycles to 40 cycles, from about 20 cycles to 40cycles, from about 20 cycles to 38 cycles, from about 20 cycles to 35cycles, from about 10 cycles to 35 cycles, from about 10 cycles to 30cycles, from about 25 cycles to 30 cycles, from about 20 cycles to 30cycles, from about 4 cycles to 8 cycles, or from about 28 cycles to 32cycles. In some embodiments, the reaction is heated to 95° C. for 3minutes before the PCR cycle begins. In some embodiments, each PCR cyclecomprises 95° C. for 3 seconds and 62° C. for 20 seconds. In someembodiments, each PCR cycle comprises 95° C. for 3 seconds, 54° C. for10 seconds, and 64° C. for 20 seconds. In some embodiments, each PCRcycle comprises 95° C. for 3 seconds and 64° C. for 20 seconds. In someembodiments, each PCR cycle comprises 95° C. for 3 seconds and 62° C.for 60 seconds. In some embodiments, each PCR cycle comprises 95° C. for3 seconds, 54° C. for 10 seconds, and 64° C. for 10 seconds. In someembodiments, the PCR comprises 30 cycles. In some embodiments, thereaction is heated to 68° C. after the completion of the PCR cycles. Insome embodiments, the reaction is heated to 68° C. from about 1 secondto about 5 seconds, from about 1 second to about 5 minutes, from about 1minute to about 5 minutes after the completion of the PCR cycles. Insome embodiments, the PCR methods described herein comprises anextension or elongation step that is at least about 5 seconds long, atleast about 6 seconds long, at least about 7 seconds long, at leastabout 8 seconds long, at least about 9 seconds long, at least about 10seconds long, at least about 11 seconds long, at least about 12 secondslong, at least about 13 seconds long, at least about 14 seconds long, atleast about 15 seconds long, at least about 20 seconds long, at leastabout 30 seconds long, at least about 40 seconds long, at least about 50seconds long, at least about 60 seconds long, at least about 90 secondslong, at least about 120 seconds long, at least about 150 seconds long,at least about 180 seconds long, at least about 210 seconds long, atleast about 240 seconds long, at least about 270 seconds long, at leastabout 300 seconds long, at least about 330 seconds long, at least about360 seconds long, at least about 390 seconds long, or more.

The time for which amplification yields a detectable amount of amplifiedproduct indicative of the presence of a target nucleic acid amplifiedcan vary depending upon the biological sample from which the targetnucleic acid was obtained, the particular nucleic acid amplificationreactions to be conducted, and the particular number of cycles ofamplification reaction desired. In some embodiments, amplification of atarget nucleic acid may yield a detectable amount of amplified productindicative to the presence of the target nucleic acid at time period of120 minutes or less; 90 minutes or less; 60 minutes or less; 50 minutesor less; 45 minutes or less; 40 minutes or less; 35 minutes or less; 30minutes or less; 25 minutes or less; 20 minutes or less; 15 minutes orless; 10 minutes or less; or 5 minutes or less.

In some embodiments, a biological sample may be preheated prior toconducting a primer extension reaction. The temperature (e.g., apreheating temperature) at which and duration (e.g., a preheatingduration) for which a biological sample is preheated may vary dependingupon, for example, the particular biological sample being analyzed. Insome examples, a biological sample may be preheated for no more thanabout 60 minutes, 50 minutes, 40 minutes, 30 minutes, 25 minutes, 20minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds.In some examples, a biological sample may be preheated at a temperaturefrom about 80° C. to about 110° C. In some examples, a biological samplemay be preheated at a temperature from about 90° C. to about 100° C. Insome examples, a biological sample may be preheated at a temperaturefrom about 90° C. to about 97° C. In some examples, a biological samplemay be preheated at a temperature from about 92° C. to about 95° C. Insome embodiments, a biological sample may be preheated at a temperatureof about 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C.,88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C.,97° C., 98° C., 99° C., or 100° C.

In some embodiments, reagents necessary for conducting nucleic acidamplification may also include a reporter agent that yields a detectablesignal whose presence or absence is indicative of the presence of anamplified product. The intensity of the detectable signal may beproportional to the amount of amplified product. In some cases, whereamplified product is generated of a different type of nucleic acid thanthe target nucleic acid initially amplified, the intensity of thedetectable signal may be proportional to the amount of target nucleicacid initially amplified. For example, in the case of amplifying atarget RNA via parallel reverse transcription and amplification of theDNA obtained from reverse transcription, reagents necessary for bothreactions may also comprise a reporter agent, may yield a detectablesignal that is indicative of the presence of the amplified DNA product,and/or the target RNA amplified. The intensity of the detectable signalmay be proportional to the amount of the amplified DNA product and/orthe original target RNA amplified. The use of a reporter agent alsoenables real-time amplification methods, including real-time PCR for DNAamplification.

Reporter agents may be linked with nucleic acids, including amplifiedproducts, by covalent or non-covalent linkages or interactions.Non-limiting examples of non-covalent linkates or interactions includeionic interactions, Van der Waals forces, hydrophobic interactions,hydrogen bonding, and combinations thereof. In some embodiments,reporter agents may bind to initial reactants and changes in reporteragent levels may be used to detect amplified product. In someembodiments, reporter agents may only be detectable (or non-detectable)as nucleic acid amplification progresses. In some embodiments, anoptically-active dye (e.g., a fluorescent dye) may be used as may beused as a reporter agent. Non-limiting examples of dyes include SYBRgreen, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidiumbromide, acridines, proflavine, acridine orange, acriflavine,fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D,chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin,phenanthridines and acridines, ethidium bromide, propidium iodide,hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidiummonoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI,acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine,SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3,TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3,BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1,YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBRGreen I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45(blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25(green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59,-61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate(FITC), tetramethyl rhodamine isothiocyanate (TRITC), rhodamine,tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5,Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), Sybr Green I, SybrGreen II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I,ethidium homodimer II, ethidium homodimer III, ethidium bromide,umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin,methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow,cascade blue, dichlorotriazinylamine fluorescein, dansyl chloride,fluorescent lanthanide complexes such as those including europium andterbium, carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein(FAM), 5- (or 6-) iodoacetamidofluorescein, 5-{[2 (and3)-5-(Acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein),lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine(ROX), 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acidtrisodium salt, 3,6-Disulfonate-4-amino-naphthalimide,phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568,594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350,405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or otherfluorophores.

In some embodiments, a reporter agent may be a sequence-specificoligonucleotide probe that is optically active when hybridized with anamplified product. Due to sequence-specific binding of the probe to theamplified product, use of oligonucleotide probes can increasespecificity and sensitivity of detection. A probe may be linked to anyof the optically-active reporter agents (e.g., dyes) and may alsoinclude a quencher capable of blocking the optical activity of anassociated dye. Non-limiting examples of probes that may be useful usedas reporter agents include TaqMan probes, TaqMan Tamara probes, TaqManMGB probes, or Lion probes. In some embodiments, a reporter agent may bea radioactive species. Non-limiting examples of radioactive speciesinclude 14C, 123I, 124I, 125I, 131I, 99mTc, 35S, or 3H. In someembodiments, a reporter agent may be an enzyme that is capable ofgenerating a detectable signal. Detectable signal may be produced byactivity of the enzyme with its substrate or a particular substrate inthe case the enzyme has multiple substrates. Non-limiting examples ofenzymes that may be used as reporter agents include alkalinephosphatase, horseradish peroxidase, I2-galactosidase, alkalinephosphatase, β-galactosidase, acetylcholinesterase, and luciferase.

In some embodiments, an amplified product (e.g., amplified DNA product,amplified RNA product) may be detected. Detection of amplified product,including amplified DNA, may be accomplished with any suitable detectionmethod. The particular type of detection method used may depend, forexample, on the particular amplified product, the type of reactionvessel used for amplification, other reagents in a reaction mixture,whether or not a reporter agent was included in a reaction mixture, andif a reporter agent was used, the particular type of reporter agent use.Non-limiting examples of detection methods include optical detection,spectroscopic detection, electrostatic detection, electrochemicaldetection, and the like. Optical detection methods include, but are notlimited to, fluorimetry and UV-vis light absorbance. Spectroscopicdetection methods include, but are not limited to, mass spectrometry,nuclear magnetic resonance (NMR) spectroscopy, and infraredspectroscopy. Electrostatic detection methods include, but are notlimited to, gel based techniques, such as, for example, gelelectrophoresis, SDS-PAGE gel. Electrochemical detection methodsinclude, but are not limited to, electrochemical detection of amplifiedproduct after high-performance liquid chromatography separation of theamplified products.

In some embodiments, the time required to complete the elements of amethod may vary depending upon the particular steps of the method. Insome embodiments, an amount of time for completing the elements of amethod may be from about 5 minutes to about 120 minutes. In someembodiments, an amount of time for completing the elements of a methodmay be from about 5 minutes to about 60 minutes. In some embodiments, anamount of time for completing the elements of a method may be from about5 minutes to about 30 minutes. In some embodiments, an amount of timefor completing the elements of a method may be less than or equal to 120minutes, less than or equal to 90 minutes, less than or equal to 75minutes, less than or equal to 60 minutes, less than or equal to 45minutes, less than or equal to 40 minutes, less than or equal to 35minutes, less than or equal to 30 minutes, less than or equal to 25minutes, less than or equal to 20 minutes, less than or equal to 15minutes, less than or equal to 10 minutes, or less than or equal to 5minutes.

In some embodiments, the reaction may have a pH suitable for producingthe product, for primer extension, protein expression, PCRamplification, or template jumping. In some embodiments, the pH of thereaction may range from about 5 to about 9, from about 6 to about 9,from about 7 to about 9, from about 8 to about 9. In some embodiments,the pH range is from about pH 2 to about pH 10, from about pH 4 to aboutpH 10, from about pH 2 to about pH 8, from about pH 4 to about pH 8,from about pH 5 to about pH 8, from about pH 5 to about pH 7, from aboutpH 6 to about pH 11, from about pH 6 to about pH 12, from about pH 5 topH 13, from about pH 5 to about pH 14. In some embodiments, the pH isabout 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0,about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11, about11.5, about 12, about 12.5, about 13, about 13.5, about 14.

In some embodiments, any method of the present disclosure may comprise adetergent. In some embodiments, the detergent is non-ionic and/or azwitterionic detergent. In some embodiments, a non-ionic detergent isselected from a group consisting of tween, triton, Triton CF-21, TritonCF-32, Triton DF-12, Triton DF-16, Triton GR-SM, Triton N-101(Polyoxyethylene branched nonylphenyl ether), Triton QS-15, TritonQS-44, Triton RW-75 (Polyethylene glycol 260 monoChexadecyl/octadecyl)ether and 1-Octadecanol), Triton X-100 (Polyethylene glycoltert-octylphenyl ether), Triton X-102, Triton X-15, Triton X-151, TritonX-200, Triton X-207, Triton X-114, Triton X-165, Triton X-305, TritonX-405 (polyoxyethylene(40) isooctylphenyl ether), Triton X-405 reduced(polyoxyethylene(40) isooctylcyclohexyl ether), Triton X-45(Polyethylene glycol 4-tert-octylphenyl ether), Triton X-705-70, TWEENin any form including: TWEEN 20 (Polyoxyethylene sorbitan monolaurate),TWEEN 21 (Polyoxyethylene sorbitan monolaurate), TWEEN 40(polyoxyethylene(20) sorbitan monopalmitate), TWEEN 60 (Polyethyleneglycol sorbitan monostearate), TWEEN 61 (Polyethylene glycol sorbitanmonostearate), TWEEN 65 (Polyoxyethylene sorbitan Tristearate), TWEEN 80(Polyoxyethylene sorbitan monooleate), TWEEN 81 (Polyoxyethylenesorbitan monooleate), TWEEN 85 (polyoxyethylene(20) sorbitan trioleate),Brij, Brij 30 (Polyoxyethylene 4 lauryl ether) Brij 35 (Polyoxyethylene23 lauryl ether), Brij 52 (Polyoxyethylene 2 cetyl ether), Brij56(Polyoxyethylene 10 cetyl ether), Brij 58 (Polyoxyethylene 20 cetylether), Brij 72 (Polyoxyethylene 2 stearyl ether), Brij 76(Polyoxyethylene 10 stearyl ether), Brij 78 (Polyoxyethylene 20 stearylether), Brij 92 (Polyoxyethylene 2 oleyl ether), Brij 97(Polyoxyethylene 10 oleyl ether), Brij 98 (Polyoxyethylene 20 oleylether), Brij700 (Polyoxyethylene 100 stearyl ether, octyl thioglucoside,maltosides, and combinations thereof.

In some embodiments, any method disclosed herein for producing anymolecule according to the present disclosure comprises at least onesalt. In some embodiments, the salt is at least one member selected fromthe group consisting of NaCl, LiCl, AlCl₃, CuCl₂, MgCl₂, InCl₃, SnCl₄,CrCl₂, CrCl₃, KCl, NaI, KI, TMACl (tetramethyl ammonium chloride), TEACl(tetraethyl ammonium chloride), KSCN, CsSCN, KCH₃COO, CH₃COONa,C₅H₈KNO₄, C₅H₈NNaO₄, CsCl, and any combination thereof. In someembodiments, any method disclosed herein for producing any moleculeaccording to the present disclosure comprises NaCl. In some embodiments,the conditions sufficient for producing a molecule or a librarycomprises NaCl. In some embodiments, the reaction may have a saltconcentration and/or NaCl suitable for producing a product, for primerextension, protein expression, PCR amplification, or template jumping.In some embodiments, the NaCl concentration is from about 50 mM to about1000 mM, from about 100 mM to about 500 mM, from about 200 mM to about300 mM, from about 200 mM to about 600 mM. In some embodiments, the NaClconcentration is at least about, at most about, or about 50 mM, at leastabout, at most about, or about 100 mM, at least about, at most about, orabout 150 mM, at least about, at most about, or about 200 mM, at leastabout, at most about, or about 250 mM, at least about, at most about, orabout 300 mM, at least about, at most about, or about 350 mM, at leastabout, at most about, or about 400 mM, at least about, at most about, orabout 450 mM, at least about, at most about, or about 500 mM, at leastabout, at most about, or about 550 mM, at least about, at most about, orat least about, at most about, or about 600 mM, at least about, at mostabout, or about 650 mM, at least about, at most about, or about 700 mM,at least about, at most about, or about 750 mM, at least about, at mostabout, or about 800 mM, at least about, at most about, or about 850 mM,at least about, at most about, or about 900 mM, at least about, at mostabout, or about 950 mM, or at least about, at most about, or about 1000mM. In some embodiments, the NaCl may improve enzyme activity and/ortemplate jumping of an enzyme or polypeptide of the present disclosure(e.g., of a reverse transcriptase).

In some embodiments, the reaction may have a nucleotide (e.g. dNTPs)concentration suitable for producing a product, for primer extension,protein expression, PCR amplification, or template jumping. In someembodiments, the total dNTP concentration in a reaction may be fromabout 50 μM to about 1000 μM, from about 100 μM to about 500 μM, fromabout 200 μM to about 300 μM, from about 200 μM to about 600 μM. In someembodiments, the total dNTP concentration is at least about, at mostabout, or about 50 μM, at least about, at most about, or about 100 μM,at least about, at most about, or about 150 μM, at least about, at mostabout, or about 200 μM, at least about, at most about, or about 250 μM,at least about, at most about, or about 300 μM, at least about, at mostabout, or about 350 μM, at least about, at most about, or about 400 μM,at least about, at most about, or about 450 μM, at least about, at mostabout, or about 500 μM, at least about, at most about, or about 550 μM,at least about, at most about, or at least about, at most about, orabout 600 μM, at least about, at most about, or about 650 μM, at leastabout, at most about, or about 700 μM, at least about, at most about, orabout 750 μM, at least about, at most about, or about 800 μM, at leastabout, at most about, or about 850 μM, at least about, at most about, orabout 900 μM, at least about, at most about, or about 950 μM, or atleast about, at most about, or about 1000 μM. In some embodiments, thetotal concentration of each dNTP is at least about, at most about, orabout 1 μM; at least about, at most about, or about 2 μM; at leastabout, at most about, or about 3 μM; at least about, at most about, orabout 4 μM; at least about, at most about, or about 5 μM; at leastabout, at most about, or about 6 μM; at least about, at most about, orabout 7 μM; at least about, at most about, or about 8 μM; at leastabout, at most about, or about 9 μM; at least about, at most about, orabout 10 μM; at least about, at most about, or about 15 μM; at leastabout, at most about, or about 20 μM; at least about, at most about, orabout 25 μM; at least about, at most about, or about 30 μM; at leastabout, at most about, or about 35 μM; at least about, at most about, orabout 40 μM; at least about, at most about, or about 45 μM; at leastabout, at most about, or about 50 μM; at least about, at most about, orabout 55 μM; at least about, at most about, or about 60 μM; at leastabout, at most about, or about 65 μM; at least about, at most about, orabout 70 μM; at least about, at most about, or about 75 μM; at leastabout, at most about, or about 80 μM; at least about, at most about, orabout 85 μM; at least about, at most about, or about 90 μM; at leastabout, at most about, or about 95 μM; at least about, at most about, orabout 100 μM; at least about, at most about, or about 250 μM; at leastabout, at most about, or about 500 μM; at least about, at most about, orabout 1000 μM; at least about, at most about, or about 10000 μM. In someembodiments, the total concentration of each dNTP is from about 2 μM toabout 5 μM, from about 2 μM to about 10 μM, from about 2 μM to about 20μM, from about 2 μM to about 50 μM, from about 2 μM to about 100 μM,from about 2 μM to about 250 μM, from about 5 μM to about 10 μM, fromabout 5 μM to about 50 μM, from about 5 μM to about 250 μM, from about 5μM to about 1000 μM.

In some embodiments, the concentration of each dNTP may be independentand different from the concentration of one or more dNTP. In someembodiments, the concentration of each dNTP for example theconcentration of each dCTP, dGTP, dTTP, or dATP may be independent anddifferent from the concentration of at least one other dNTP. In someembodiments, the concentration of one dNTP (e.g., dCTP, dGTP, dTTP, ordATP) may be at least about or at most about or about 1 fold, 2 fold, 3fold, 4 fold, 5 fold, 7 fold, 10 fold, 20 fold, 35 fold, 50 fold, 75fold, 90 fold, 100 fold, 200 fold, 500 fold, or 1000 fold different fromat least one other dNTP (e.g., dCTP, dGTP, dTTP, or dATP).

In some embodiments, the reaction mixture includes a pH adjusting agent.pH adjusting agents of interest include, but are not limited to, sodiumhydroxide, hydrochloric acid, phosphoric acid buffer solution, trisbuffer, citric acid buffer solution, and the like. For example, the pHof the reaction mixture can be adjusted to the desired range by addingan appropriate amount of the pH adjusting agent.

The temperature range suitable for production of a product may varyaccording to factors such as the particular polymerase employed, themelting temperatures of any optional primers employed, etc. In someembodiments, the polymerase may include, but it is not limited to, areverse transcriptase, a Moloney Murine Leukemia Virus (MMLV) reversetranscriptase, an R2 reverse transcriptase, an RNA-directed DNApolymerase, an DNA-directed DNA polymerase, a non-LTR retrotransposon,an R2 non-LTR retrotransposon, a polypeptide having reversetranscriptase activity, or any variant thereof, or any combinationthereof.

In some embodiments, the conditions sufficient to produce a productinclude bringing the reaction mixture to a temperature ranging fromabout 4° C. to about 72° C., from about 16° C. to about 70° C., fromabout 37° C. to about 50° C., from about 40° C. to about 45° C., fromabout 30° C. to about 42° C., from about 25° C. to about 42° C., fromabout 25° C. to about 30° C., from about 28° C. to about 32° C., fromabout 29° C. to about 31° C. In some embodiments, the temperature isabout 15° C., about 16° C., about 17° C., about 18° C., about 19° C.,about 20° C., about 21° C., about 22° C., about 23° C., about 24° C.,about 25° C., about 26° C., about 27° C., about 28° C., about 29° C.,about 30° C., about 31° C., about 32° C., about 33° C., about 34° C.,about 35° C., about 36° C., about 37° C., about 38° C., about 39° C.,about 40° C., about 41° C., about 42° C., about 43° C., about 44° C.,about 45° C., about 46° C., about 47° C., about 48° C., about 49° C.,about 50° C., about 51° C., about 52° C., about 53° C., about 54° C.,about 55° C., about 56° C., about 57° C., about 58° C., about 59° C.,about 60° C., about 61° C., about 62° C., about 63° C., about 64° C.,about 65° C., about 66° C., about 67° C., about 68° C., about 69° C.,about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., orabout 75° C. In some embodiments, the temperature is about or at mostabout 42° C. In some embodiments, the temperature is about or at mostabout 50° C. In some embodiments, the temperature is about or at mostabout 35° C. In some embodiments, the temperature is about or at mostabout 25° C. In some embodiments, the temperature is about or at mostabout 30° C. In some embodiments, the reaction is incubated from about20 minutes to about 3 hours, from about 30 minutes to about 1.5 hours,from about 30 minutes to about 1 hour, from about 30 minutes to about 2hours, from about 1 hour to about 2 hours, from about 1 hour to about1.5 hours, from about 30 minutes to about 5 hours, from about 1 hour toabout 3 hours, from about 1 hour to about 4 hours, from about 1 hour toabout 5 hours. In some embodiments, the reaction is incubated for about1 hour. In some embodiments, the reaction is incubated for about 20minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours,about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours. Insome embodiments, the reaction is incubated for at least at least about20 minutes, at least about 30 minutes, at least about 40 minutes, atleast about 50 minutes, at least about 1 hour, at least about 1.5 hours,at least about 2 hours, at least about 2.5 hours, at least about 3hours, at least about 3.5 hours, at least about 4 hours, at least about4.5 hours, or at least about 5 hours. In some embodiments, the reactionis incubated at about 30° C. for about 1 hour, or at about 42° C. forabout 1 hour. In some embodiments, the conditions sufficient forgenerating a molecule or a nucleic acid molecule comprises a temperatureof about 12° C. to about 42° C. for about 1 minute to about 5 hours. Insome embodiments, the conditions sufficient for generating a molecule ora nucleic acid molecule comprises a temperature of about 8° C. to about50° C. for about 1 minute to about 24 hours.

In some embodiments, a primer can be designed to be a certain length. Insome embodiments, a primer can be from about 6 to about 100 nucleotides,from about 6 to about 90 nucleotides, from about 6 to about 80nucleotides, from about 6 to about 70 nucleotides, from about 6 to about60 nucleotides, from about 6 to about 50 nucleotides, from about 6 toabout 40 nucleotides, from about 6 to about 30 nucleotides, from about 6to about 20 nucleotides, or from about 6 to about 10 nucleotides inlength. In some embodiments, a primer can be from about 25 to about 80,from about 25 to about 75, from about 25 to about 70, from about 25 toabout 65, from about 25 to about 60, from about 25 to about 55, fromabout 25 to about 50, from about 25 to about 45, from about 25 to about40, from about 25 to about 35, or from about 25 to about 30 bases inlength. In some embodiments, a primer can be at least about 5, at leastabout 6, at least about 7, at least about 8, at least about 9, at leastabout 10, at least about 11, at least about 12, at least about 13, atleast about 14, at least about 15, at least about 16, at least about 17,at least about 18, at least about 19, at least about 20, at least about25, at least about 30, at least about 35, at least about 40, at leastabout 45, at least about 50, at least about 55, at least about 60, atleast about 65, at least about 70, at least about 75, at least about 80,at least about 85, at least about 90, at least about 95 or at leastabout 100 bases in length. In some embodiments, a primer can be about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 95 or about 100 bases in length. In some embodiments, a primercan be at least about, no more than about, or about 120, 130, 140, 150,160, 170, 180, 190, 200, 230, 250, 270, 290, 300, 320, 340, 350, 370,400, 420, 450, 470, 490, or 500.

In some embodiments, a primer can be designed to anneal to a target at agiven melting temperature (Tm). In some embodiments, a Tm can be fromabout 20° C. to about 100° C., about 20° C. to about 90° C., about 20°C. to about 80° C., about 20° C. to about 70° C., about 20° C. to about60° C., about 20° C. to about 50° C., about 20° C. to about 40° C., orabout 20° C. to about 30° C. In some embodiments, a Tm can be at leastabout, at most about, or about 20° C., 21° C., 22° C., 23° C., 24° C.,25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C.,34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C.,43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C.,52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C.,61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C.,70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C.,79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C.,88° C., 89° C., 90° C., 91° C., 92° C., 83° C., 84° C., 85° C., 96° C.,97° C., 98° C., 99° C., or 100° C. A plurality of primers can bedesigned to have Tms within a range, e.g., within a range spanning 15°C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° C., 2° C., or1° C. A plurality of primers can be designed to have identical Tms.

In some embodiments the enzyme, or modified enzyme (e.g., modifiedreverse transcriptase), or protein, or polypeptide, or a variant, or aPCR product, or a cDNA molecule, or a template, or a nucleic acidmolecule, or any component of the present disclosure may be purified. Insome embodiments, the fragmented or degraded nucleic acid (e.g., RNA orDNA) may be purified. In some embodiments, the reverse transcriptase ora modified reverse transcriptase may be purified. In some embodiments,the R2 reverse transcriptase or a modified R2 reverse transcriptase maybe purified. In some embodiments, the non-LTR retrotransposon protein orpolypeptide having reverse transcriptase activity, or a modified non-LTRretrotransposon protein or a modified polypeptide having reversetranscriptase activity may be further purified. In some embodiments, thecDNA molecule may be purified. In some embodiments, the template may bepurified. In some embodiments, the acceptor nucleic acid molecule may bepurified.

Purification may comprise precipitation, ultracentrifugation,chromatographic method based on size, charge, hydrophobicity, affinity,metal binding, HPLC. In some embodiments, the purification comprisescolumn chromatography. In some embodiments, the column chromatographymay be size exclusion (SEC), ion exchange (IEX), affinitychromatography, immobilized metal ion affinity chromatography (IMAC),Ni-IMAC chromatography, and/or hydrophobic interaction (HIC). In someembodiments, the purification comprises His-tag affinity resin. In someembodiments, the purification may comprise one step. In someembodiments, the purification may comprise two steps. In someembodiments, the two step purification comprises nickel and heparin. Insome embodiments, the two step purification comprises nickel and heparinaffinity purifications. In some embodiments, the two purification stepsprovide higher activity and/or increased template jumping compared toone step purification. In some embodiments, the purification comprisesheparin-affinity purification. In some embodiments, purification mayinclude affinity purification, Ni-NTA affinity, fast protein liquidchromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems),high-pressure liquid chromatography (HPLC) (e.g., Beckman and WatersHPLC). In some embodiments, purification may include, but not limitedto, ion exchange chromatography (e.g., Q, S), size exclusionchromatography, salt gradients, affinity purification (e.g., Ni, Co,FLAG, maltose, glutathione, protein A/G), gel filtration, reverse-phase,ceramic HYPERD (Registered trademark) ion exchange chromatography, andhydrophobic interaction columns (HIC). Also included are analyticalmethods such as SDS-PAGE (e.g., coomassie, silver stain), immunoblot,Bradford, and ELISA, which may be utilized during any step of theproduction or purification process, typically to measure the purity ofthe protein or enzyme composition.

In some embodiments, the overall activity of the purified enzyme,protein, polypeptide, the R2 reverse transcriptase, the non-LTRretrotransposon protein or polypeptide having reverse transcriptaseactivity, the reverse transcriptase, or variants thereof, or productsthereof using a two-step purification is at least about 2%, at leastabout 5%, at least about 7%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99% higher than the overall activity using the one-step purification. Insome embodiments, the overall activity of the purified enzyme, protein,polypeptide, the R2 reverse transcriptase, the non-LTR retrotransposonprotein or polypeptide having reverse transcriptase activity, thereverse transcriptase, or variants thereof, or products thereof is atleast about 0.5%, at least about 1%, at least about 1.5%, at least about2%, at least about 5%, at least about 7%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99% higher than the overall activity of the non-purifiedenzyme, protein, polypeptide, R2 reverse transcriptase, non-LTRretrotransposon protein or polypeptide having reverse transcriptaseactivity, reverse transcriptase, or variants thereof, or productsthereof. In some embodiments, a purified enzyme, protein, polypeptide,R2 reverse transcriptase, the non-LTR retrotransposon protein, orpolypeptide having reverse transcriptase activity, reversetranscriptase, modified enzyme, modified reverse transcriptase, modifiedpolypeptide having reverse transcriptase activity, or variants thereof,or products thereof is at least about 0.5%, at least about 1%, at leastabout 3%, at least about or about 5%, at least about or about 10%, atleast about or about 15%, at least about or about 20%, at least about orabout 25%, at least about or about 30%, at least about or about 35%, atleast about or about 40%, at least about or about 45%, at least about orabout 50%, at least about or about 55%, at least about or about 60%, atleast about or about 61%, at least about or about 62%, at least about orabout 63%, at least about or about 64%, at least about or about 65%, atleast about or about 66%, at least about or about 67%, at least about orabout 68%, at least about or about 69%, at least about or about 70%, atleast about or about 71%, at least about or about 72%, at least about orabout 73%, at least about or about 74%, at least about or about 75%, atleast about or about 76%, at least about or about 77%, at least about orabout 78%, at least about or about 79%, at least about or about 80%, atleast about or about 81%, at least about or about 82%, at least about orabout 83%, at least about or about 84%, at least about or about 85%, atleast about or about 86%, at least about or about 87%, at least about orabout 88%, at least about or about 89%, at least about or about 90%, atleast about or about 91%, at least about or about 92%, at least about orabout 93%, at least about or about 94%, at least about or about 95%, atleast about or about 96%, at least about or about 97%, at least about orabout 98%, or at least about or about 99% pure.

In some embodiments, the purified enzyme, protein, polypeptide, R2reverse transcriptase, non-LTR retrotransposon protein or polypeptidehaving reverse transcriptase activity, reverse transcriptase, orvariants thereof, or products thereof produces template jumping that isat least about or about one time, at least about or about two times, atleast about or about three times, at least about or about four times, atleast about or about five times, at least about or about six times, atleast about or about seven times, at least about or about eight times,at least about or about nine times, at least about or about ten times,at least about or about fifteen times, at least about or about twentytimes, at least about or about twenty five times, at least about orabout thirty times, at least about or about forty times, at least aboutor about fifty times, at least about or about seventy times, at leastabout or about eighty times, at least about or about ninety times, atleast about or about 100 times, at least about or about 150 times, atleast about or about 200 times, at least about or about 250 times, atleast about or about 300 times, at least about or about 350 times, atleast about or about 400 times, at least about or about 500 times, atleast about or about 700 times, at least about or about 1000 times, atleast about or about 10000 times more and/or higher intensity than thenon-purified enzyme, protein, polypeptide, R2 reverse transcriptase,non-LTR retrotransposon protein or polypeptide having reversetranscriptase activity, reverse transcriptase, or variants thereof, orproducts thereof.

Cell-Free DNA and Circulating Tumor DNA

In some embodiments, obtaining a quantity of circulating tumor (ctDNA)may comprise PCR. In some embodiments, obtaining a quantity of ctDNA maycomprise digital PCR. In some embodiments, obtaining a quantity of ctDNAmay comprise quantitative PCR. In some embodiments, obtaining a quantityof ctDNA may comprise obtaining sequencing information on the ctDNA. Thesequencing information may comprise information relating to one or moregenomic regions based. In some embodiments, obtaining the quantity ofctDNA may comprise hybridization of the ctDNA to an array.

Determining the quantity of the cell-free DNA may comprise determiningabsolute quantities of the cell-free DNA. The quantity of the cell-freeDNA may be determined by counting sequencing reads pertaining to thecell-free DNA. The quantity of the cell-free DNA may be determined byquantitative PCR.

Determining quantities of cell-free DNA (cf DNA) may be performed bymolecular barcoding of the cfDNA. Molecular barcoding of the cf DNA maycomprise attaching adaptors to one or more ends of the cf DNA. Theadaptor may comprise a plurality of oligonucleotides. The adaptor maycomprise one or more deoxyribonucleotides. The adaptor may compriseribonucleotides. The adaptor may be single-stranded. The adaptor may bedouble-stranded. The adaptor may comprise double-stranded andsingle-stranded portions. For example, the adaptor may be a Y-shapedadaptor. The adaptor may be a linear adaptor. The adaptor may be acircular adaptor. The adaptor may comprise a molecular barcode, sampleindex, primer sequence, linker sequence or a combination thereof. Themolecular barcode may be adjacent to the sample index. The molecularbarcode may be adjacent to the primer sequence. The sample index may beadjacent to the primer sequence. A linker sequence may connect themolecular barcode to the sample index. A linker sequence may connect themolecular barcode to the primer sequence. A linker sequence may connectthe sample index to the primer sequence.

The adaptor may comprise a molecular barcode. The molecular barcode maycomprise a random sequence. The molecular barcode may comprise apredetermined sequence. Two or more adaptors may comprise two or moredifferent molecular barcodes. The molecular barcodes may be optimized tominimize dimerization. The molecular barcodes may be optimized to enableidentification even with amplification or sequencing errors. Forexamples, amplification of a first molecular barcode may introduce asingle base error. The first molecular barcode may comprise greater thana single base difference from the other molecular barcodes. Thus, thefirst molecular barcode with the single base error may still beidentified as the first molecular barcode. The molecular barcode maycomprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Themolecular barcode may comprise at least 3 nucleotides. The molecularbarcode may comprise at least 4 nucleotides. The molecular barcode maycomprise less than 20, 19, 18, 17, 16, or 15 nucleotides. The molecularbarcode may comprise less than 10 nucleotides. The molecular barcode maycomprise less than 8 nucleotides. The molecular barcode may compriseless than 6 nucleotides. The molecular barcode may comprise 2 to 15nucleotides. The molecular barcode may comprise 2 to 12 nucleotides. Themolecular barcode may comprise 3 to 10 nucleotides. The molecularbarcode may comprise 3 to 8 nucleotides. The molecular barcode maycomprise 4 to 8 nucleotides. The molecular barcode may comprise 4 to 6nucleotides.

The adaptor may comprise a sample index. The sample index may comprise arandom sequence. The sample index may comprise a predetermined sequence.Two or more sets of adaptors may comprise two or more different sampleindexes. Adaptors within a set of adaptors may comprise identical sampleindexes. The sample indexes may be optimized to minimize dimerization.The sample indexes may be optimized to enable identification even withamplification or sequencing errors. For examples, amplification of afirst sample index may introduce a single base error. The first sampleindex may comprise greater than a single base difference from the othersample indexes. Thus, the first sample index with the single base errormay still be identified as the first molecular barcode. The sample indexmay comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.The sample index may comprise at least 3 nucleotides. The sample indexmay comprise at least 4 nucleotides. The sample index may comprise lessthan 20, 19, 18, 17, 16, or 15 nucleotides. The sample index maycomprise less than 10 nucleotides. The sample index may comprise lessthan 8 nucleotides. The sample index may comprise less than 6nucleotides. The sample index may comprise 2 to 15 nucleotides. Thesample index may comprise 2 to 12 nucleotides. The sample index maycomprise 3 to 10 nucleotides. The sample index may comprise 3 to 8nucleotides. The sample index may comprise 4 to 8 nucleotides. Thesample index may comprise 4 to 6 nucleotides.

The adaptor may comprise a primer sequence. The primer sequence may be aPCR primer sequence. The primer sequence may be a sequencing primer.

Adaptors may be attached to one end of the cf DNA. Adaptors may beattached to both ends of the cf DNA. Adaptors may be attached to one ormore ends of a single-stranded cf DNA. Adaptors may be attached to oneor more ends of a double-stranded cfDNA.

Adaptors may be attached to the cf DNA by ligation. Ligation may beblunt end ligation. Ligation may be sticky end ligation. Adaptors may beattached to the cf DNA by primer extension. Adaptors may be attached tothe cf DNA by reverse transcription. Adaptors may be attached to the cfDNA by hybridization. Adaptors may comprise a sequence that is at leastpartially complementary to the cf DNA. Alternatively, in some instances,adaptors do not comprise a sequence that is complementary to the cf DNA.

The cf-DNA may be derived from a tumor in the subject. The cf-DNA may bederived from any sample of the subject. The cf-DNA may be derived fromany organ of the subject. The cf-DNA may be derived from any liquid ofthe subject (e.g., blood, saliva, urine, and mucus). The method mayfurther comprise detecting a cancer in the subject based on thedetection of the cf-DNA.

In some embodiments, the presence or absence of a sequence can be linkedto a DNA or RNA profile or to a cancer. For example, the presence orabsence of a sequence, can be linked to a transcription profile,microRNA profile, cancer profile, or genomic mutation profile of asample, such as a single cell. In some embodiments, cfDNA can be usedfor cancer profiling, and/or monitoring a condition progression, and/ormonitoring the occurrence of previous and new mutations. The method mayfurther comprise diagnosing a cancer in the subject based on thedetection of the cf-DNA. Diagnosing the cancer may have a sensitivity ofat least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%,77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or99%. Diagnosing the cancer may have a specificity of at least about 50%,52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%,87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. The method mayfurther comprise prognosing a cancer in the subject based on thedetection of the cf-DNA. Prognosing the cancer may have a sensitivity ofat least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%,77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or99%. Prognosing the cancer may have a specificity of at least about 50%,52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%,87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. The method mayfurther comprise determining a therapeutic regimen for the subject basedon the detection of the cf-DNA. The method may further compriseadministering an anti-cancer therapy to the subject based on thedetection of the cf-DNA. The method may further comprise detectingmutations (e.g., mutations at specific regions) based on the sequencinginformation. Diagnosing the cancer may be based on the detection ofmutations. The detection of at least 3 mutations may be indicative ofthe cancer. The detection of one or more mutations in three or moreregions may be indicative of the cancer.

Mutation of Enzymes

In some embodiments, a modified enzyme, or derivatives and variants maybe prepared during synthesis of the peptide or by post-productionmodification. In some embodiments, a modified enzyme, or derivatives andvariants may be produced by site-directed mutagenesis (e.g. Q5®Site-Directed Mutagenesis Kit Protocol), random mutagenesis, orenzymatic cleavage and/or ligation of nucleic acids. In someembodiments, the derivatives and variants, or a modified enzyme areproduced by random mutagenesis. In some embodiments, a rational designand/or mutagenesis is based on sequence alignment analysis. In someembodiments, the rational design/mutagenesis is based on sequencealignment analysis with defined and known enzymes and proteins. In someembodiments, sequence alignment analysis is performed with enzymesand/or elements with homology to R2, including, but not limited to,non-LTR retrotransposons, telomerase, group II introns, LTRretrotransposons, reverse transcriptase, retroviral reversetranscriptase (e.g., HIV, MMLV), and viral RNA dependent RNA polymerase.

In some embodiments, variants or modified enzymes of the presentdisclosure can be produced by, including, but not limited to, forexample, site-saturation mutagenesis, scanning mutagenesis, insertionalmutagenesis, deletion mutagenesis, random mutagenesis, site-directedmutagenesis, and directed-evolution, as well as various otherrecombinatorial approaches. Methods for making modified enzymes,polynucleotides and proteins (e.g., variants) include DNA shufflingmethodologies, methods based on non-homologous recombination of genes,such as ITCHY (See, Ostermeier et al., 7:2139-44 [1999]), SCRACHY (See,Lutz et al. 98:11248-53 [2001]), SHIPREC (See, Sieber et al., 19:456-60[2001]), and NRR (See, Bittker et al., 20:1024-9 [2001]; Bittker et al.,101:7011-6 [2004]), and methods that rely on the use of oligonucleotidesto insert random and targeted mutations, deletions and/or insertions(See, Ness et al., 20:1251-5 [2002]; Coco et al., 20:1246-50 [2002]; Zhaet al., 4:34-9 [2003]; Glaser et al., 149:3903-13 [1992]). In someembodiments, polynucleotides, polypeptides, proteins, or enzymes of thepresent disclosure may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. Polynucleotides, polypeptides, proteins,or enzymes of the present disclosure may be produced by DNA shuffling,gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. DNAshuffling may be employed to modulate the activities of polynucleotides,polypeptides, proteins, or enzymes of the present disclosure, suchmethods can be used to generate polypeptides with altered activity. See,generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252;5,837,458; and 6,444,468; and Patten et al., Curr. Opinion Biotechnol.8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998);Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo andBlasco, Biotechniques 24(2):308-13 (1998). Polynucleotides,polypeptides, proteins, or enzymes of the present disclosure may containone or more components, motifs, sections, parts, domains, fragments,etc., of a polynucleotide, polypeptide, protein, or enzyme of thepresent disclosure. In some embodiments, kits for use in mutagenic PCR,such as, for example, the Diversify PCR Random Mutagenesis Kit(Clontech) or the GeneMorph Random Mutagenesis Kit (Stratagene) may beused.

In some embodiments, variant proteins differ from a parent protein ormodified enzymes differ from a wild-type or unmodified enzyme and oneanother by a small number of amino acid residues. The number ofdiffering amino acid residues may be one or more, preferably about 1, 2,3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acidresidues. In some embodiments, the number of different amino acidsbetween variants is between about 1 and about 10. In some embodiments,related proteins and particularly variant proteins comprise at leastabout 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, or 99% amino acid sequence identity. Additionally, a relatedprotein or a variant protein as used herein, refers to a protein thatdiffers from another related protein or a parent protein in the numberof prominent regions. For example, in some embodiments, variant proteinshave about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 corresponding prominentregions that differ from the parent protein.

In some embodiments, screening methods can include conventionalscreening methods such as liquid phase, or microtiter plate basedassays. The format for liquid phase assays is often roboticallymanipulated 96, 384, or 1536-well microtiter plates. Other screeningmethods include growth selection (Snustad et al., 1988; Lundberg et al.,1993; Yano et al., 1998), colorimetric screening of bacterial coloniesor phage plaques (Kuritz, 1999), in vitro expression cloning (King etal., 1997) and cell surface or phage display (Benhar, 2001). In someembodiments, screening approaches may be a method selected fromyeast-2-hybrid, n-hybrid, reverse-2-hybrid, reverse n-hybrid, split twohybrid, bacterial display, phage display, retroviral display, ribosomedisplay, covalent display, in vitro display, or any other displaymethod. In some embodiments, the library is screened using a phagedisplay method.

Analysis of the sequences derived from template jumps: the bandcorresponding to the template jump product may be excised from apolyacrylamide gel, eluted with sodium acetate (e.g. 0.3 M sodiumacetate, pH 5.2), SDS (e.g. 0.03%) for several hours at roomtemperature, phenol/chloroform extracted and ethanol precipitated. Theisolated cDNA may then be used as a template for PCR amplification usingone or more primer(s). The PCR products may then be directly cloned intoa vector (Burke et al., “R4, a non-LTR Retrotransposon Specific to theLarge Subunit rRNA Gene of Nematodes,” Nucleic Acids Res. 23: 4628-4634(1995)) and individual clones sequenced.

Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, the kit, in a suitable container, comprises one ormore primer(s). The kit can also comprise a computer readable medium,e.g., non-transitory computer readable medium. The kit can also comprisereaction components for primer extension and amplification (e.g., dNTPs,polymerase, buffers). The kit can include reagents for library formation(e.g., primers (probes), dNTPs, polymerase, and enzymes). The kit mayalso comprise approaches for purification, such as a bead suspension.The kit can include reagents for sequencing, e.g., fluorescentlylabelled dNTPs, sequencing primers, etc.

In some embodiments, some of the components of the kit may be packagedeither in aqueous media or in lyophilized form. The containers of thekits can include at least one vial, test tube, flask, bottle, syringe orother containers, into which a component may be placed and suitablyaliquoted. Where there is more than one component in the kit, the kitalso can contain a second, third or other additional container intowhich the additional components may be separately placed. Variouscombinations of components may be comprised in a container. In someembodiments, various combinations of components may be comprised in avial. The kits of the present disclosure may also contain the componentsin close confinement for commercial sale. Such containers may includeinjection or blow molded plastic containers into which the desired vialsare retained.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution can be an aqueous solution. Thecomponents of the kit may be provided as dried powder(s). When reagentsand/or components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent.

A kit can include instructions for employing the kit components as wellthe use of any other reagent not included in the kit. Instructions mayinclude variations that can be implemented.

In some embodiments, reagents and materials include primers foramplifying desired sequences, nucleotides, suitable buffers or bufferreagents, salt, and so forth, and in some cases the reagents includeapparatus or reagents for isolation of a particular desired cell(s). Insome embodiments, there are one or more apparatuses in the kit suitablefor extracting one or more samples from an individual. The apparatus maybe a syringe, fine needles, scalpel, and so forth.

In some embodiments, a kit may be used for the preparation of cDNA froma template (e.g. RNA template). Such a kit may include a carrier devicecompartmentalized to receive one or more containers, such as vials,tubes, and the like, each of which includes one of the separate elementsused to prepare cDNA from RNA. For example, there may be provided afirst container, the contents of which include a reverse transcriptase(e.g. non-retroviral reverse transcriptase, non-LTR retrotransposon, R2reverse transcriptase) or variants thereof, in a liquid solution, powderform, or lyophilized form. Further, any number of additional containerscan be provided, the contents of which independently include suitablebuffers, substrates for nucleotide synthesis such as the deoxynucleotidetriphosphates (e. g., dATP, dCTP, dGTP, and dTTP) either individually orcollectively in a suitable solution, a template (e.g. template RNA), oneor more primer(s), and acceptor nucleic acid molecule (e.g. acceptorRNA), and optionally a terminal transferase in solution. In someembodiments, a kit may comprise a fragment or degraded nucleic acid,DNA, RNA, or a combination thereof, one of more primer(s), an acceptornucleic acid molecule (e.g., an acceptor nucleic acid moleculecomprising a modified nucleotide), a reverse transcriptase (e.g.,non-retroviral reverse transcriptase, non-LTR retrotransposon, R2reverse transcriptase) or variants thereof, suitable buffers, substratesfor nucleotide synthesis such as the deoxynucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP). Any combinations of the abovecomponents can be provided. Any of the above components may be excludedfrom the kit. In some embodiments, the one or more primer(s) may be oneor more random primer(s). In some embodiments, any of the components maybe individually packed.

Target Molecule

In some instances, the molecules are DNA, RNA, or DNA-RNA hybrids. Themolecules may be single-stranded or double-stranded. In someembodiments, the molecules are RNA molecules, such as mRNA, rRNA, tRNA,ncRNA, lncRNA, siRNA, microRNA or miRNA. The RNA molecules may bepolyadenylated. Alternatively, the mRNA molecules are notpolyadenylated. Alternatively, the molecules are DNA molecules. The DNAmolecules may be genomic DNA. The DNA molecules may comprise exons,introns, untranslated regions, or any combination thereof. In someembodiments, the molecules are a panel of molecules.

In some embodiments, the molecule is a fragment or degraded molecule. Insome embodiments, the fragment or degraded molecule is a fragment DNA,degraded DNA, fragment RNA, degraded RNA, or combinations thereof. Insome embodiments, the molecule is a template nucleic acid (e.g.,template DNA, RNA, or combinations thereof). In some embodiments, themolecule is a nucleic acid. In some embodiments, the total amount of amolecule is from about 1 femtomolar (fM) to about 100 micromolar, fromabout 40 femtomolar to about 0.01 micromolar, from about 50 femtomolarto about 500 femtomolar, from about 50 femtomolar to about 0.01micromolar, from about 50 femtomolar to about 0.1 micromolar, from about50 femtomolar to about 500 picomolar, from about 50 femtomolar to about500 nanomolar, from about 50 femtomolar to about 500 micromolar, fromabout 50 femtomolar to about 1 picomolar, from about 40 femtomolar toabout 1 nanomolar, from about 1 femtomolar to about 1 picomolar, fromabout 0.1 nM to about 100 nM. In some embodiments, the total about of amolecule is equal to or lower than about 1000 micromolar, equal to orlower than about 500 micromolar, equal to or lower than about 250micromolar, equal to or lower than about 100 micromolar, equal to orlower than about 50 micromolar, equal to or lower than about 25micromolar, equal to or lower than about 10 micromolar, equal to orlower than about 1 micromolar, equal to or lower than about 0.1micromolar, equal to or lower than about 0.01 micromolar, equal to orlower than about 0.001 micromolar, equal to or lower than about 0.0001micromolar, equal to or lower than about 2000 nanomolar, equal to orlower than about 500 nanomolar, equal to or lower than about 250nanomolar, equal to or lower than about 200 nanomolar, equal to or lowerthan about 50 nanomolar, equal to or lower than about 25 nanomolar,equal to or lower than about 20 nanomolar, equal to or lower than about2 nanomolar, equal to or lower than about 0.2 nanomolar, equal to orlower than about 0.01 nanomolar, equal to or lower than about 0.001nanomolar, equal to or lower than about 0.0001 nanomolar, equal to orlower than about 3000 picomolar, equal to or lower than about 500picomolar, equal to or lower than about 250 picomolar, equal to or lowerthan about 300 picomolar, equal to or lower than about 50 picomolar,equal to or lower than about 25 picomolar, equal to or lower than about30 picomolar, equal to or lower than about 3 picomolar, equal to orlower than about 0.3 picomolar, equal to or lower than about 0.01picomolar, equal to or lower than about 0.001 picomolar, equal to orlower than about 0.0001 picomolar, equal to or lower than about 5000femtomolar, equal to or lower than about 500 femtomolar, equal to orlower than about 250 femtomolar, equal to or lower than about 50femtomolar, equal to or lower than about 25 femtomolar, equal to orlower than about 10 femtomolar, equal to or lower than about 1femtomolar, equal to or lower than about 0.1 femtomolar, equal to orlower than about 0.01 femtomolar, equal to or lower than about 0.001femtomolar, equal to or lower than about 0.0001 femtomolar.

The methods and kits disclosed herein may be used to stochasticallylabel individual occurrences of identical or nearly identical moleculesand/or different molecules. In some instances, the methods and kitsdisclosed herein may be used to stochastically label identical or nearlyidentical molecules (e.g., molecules comprise identical or nearlyidentical sequences). For example, the molecules to be labeled compriseat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity. The nearly identical molecules may differ by less than about100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1 nucleotide or base pair. The plurality of nucleic acids in one ormore samples of the plurality of samples may comprise two or moreidentical sequences. At least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, or 100% of the total nucleic acids in one ormore of the plurality of samples may comprise the same sequence. Theplurality of nucleic acids in one or more samples of the plurality ofsamples may comprise at least two different sequences. At least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of the total nucleic acids inone or more of the plurality of samples may comprise at least twodifferent sequences. In some instances, the molecules to be labeled arevariants of each other. In some embodiments, the molecules to be labeledare fragment or degraded nucleic acid (e.g., DNA or RNA). In someembodiments, the molecules to be labeled are unknown. In someembodiments, the molecules to be labeled are in the femtomolar range,nanomolar range, micromolar range, or millimolar range. In someembodiments, the molecules to be labeled may contain single nucleotidepolymorphisms or other types of mutations. In some embodiments, themolecules to be labeled are splice variants. In some embodiments, atleast one molecule is stochastically labeled. In some embodiments, atleast about 2, 3, 4, 5, 6, 7, 8, 9, or 10 identical or nearly identicalmolecules are stochastically labeled. In some embodiments, at leastabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000 identical or nearly identical molecules arestochastically labeled. In some embodiments, at least 1500; 2,000; 2500;3,000; 3500; 4,000; 4500; 5,000; 6,000; 7,000; 8,000; 9,000; or 10000identical or nearly identical molecules are stochastically labeled. Insome embodiments, at least 15,000; 20,000; 25,000; 30,000; 35,000;40,000; 45,000; 50,000; 60,000; 70,000; 80,000; 90,000; or 100,000identical or nearly identical molecules are stochastically labeled. Insome embodiments, the one or more molecules are detected. In someembodiments, the one or more molecules are sequenced. In someembodiments, one or more unknown molecules present in the femtomolarrange is amplified (e.g. by PCR), and/or labeled, and/or detected,and/or sequenced. In some embodiments, the one or more unknown moleculesis indicative of a disease. In some embodiments, the one or more unknownmolecules present in the femtomolar range is indicative of a disease. Insome embodiments, the disease is cancer or tumor.

In some embodiments, the methods and kits disclosed herein may be usedto stochastically label different molecules. For example, the moleculesto be labeled comprise less than about 75%, 70%, 65%, 60%, 55%, 50%,45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequenceidentity. The different molecules may differ by at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 ormore nucleotides or base pairs. In some instances, at least one moleculeis stochastically labeled. In some embodiments, at least 2, 3, 4, 5, 6,7, 8, 9, or 10 different molecules are stochastically labeled. In someembodiments, at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000 different molecules arestochastically labeled. In some embodiments, at least about 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, or 10000different molecules are stochastically labeled. In some embodiments, atleast 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 60000,70000, 80000, 90000, or 100000 different molecules are stochasticallylabeled. In some embodiments, the molecules are sequenced.

In some embodiments, the different molecules to be labeled may bepresent in the sample at different concentrations or amounts. Forexample, the concentration or amount of one molecule may be greater thanthe concentration or amount of another molecule in the sample. In someembodiments, the concentration or amount of at least one molecule in thesample is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more timesgreater than the concentration or amount of at least one other moleculein the sample. In some embodiments, the concentration or amount of atleast one molecule in the sample is at least about 1000 or more timesgreater than the concentration or amount of at least one other moleculein the sample. In some embodiments, the concentration or amount of onemolecule is less than the concentration or amount of another molecule inthe sample. In some embodiments, the concentration or amount of at leastone molecule in the sample may be at least about 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, or 100 or more times less than the concentration or amount of atleast one other molecule in the sample. In some embodiments, theconcentration or amount of at least one molecule in the sample may be atleast about 1000 or more times less than the concentration or amount ofat least one other molecule in the sample.

In some embodiments, the molecules to be labeled are in one or moresamples. The molecules to be labeled may be in two or more samples. Thetwo or more samples may contain different amounts or concentrations ofthe molecules to be labeled. In some embodiments, the concentration oramount of one molecule in one sample may be greater than theconcentration or amount of the same molecule in a different sample. Insome embodiments, a blood sample may contain a higher amount of aparticular molecule than a urine sample. In some embodiments, a singlesample is divided into two or more subsamples. The subsamples maycontain different amounts or concentrations of the same molecule. Theconcentration or amount of at least one molecule in one sample may be atleast about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more times greater thanthe concentration or amount of the same molecule in another sample. Insome embodiments, the concentration or amount of one molecule in onesample may be less than the concentration or amount of the same moleculein a different sample. For example, a skin tissue sample may contain ahigher amount of a particular molecule than a lung tissue sample. Theconcentration or amount of at least one molecule in one sample may be atleast about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more times less than theconcentration or amount of the same molecule in another sample.

In some embodiments, the methods and kits disclosed herein may be usedfor the analysis of one or more molecules from two or more samples. Theone or more molecules may comprise one or more polypeptides. The methodmay comprise determining the identity of one or more labeledpolypeptides. Determining the identity of one or more labeledpolypeptides may comprise mass spectrometry. The method may furthercomprise combining the labeled polypeptides of the first sample with thelabeled polypeptides of the second sample. The labeled polypeptides maybe combined prior to determining the number of different labeledpolypeptides. The method may further comprise combining the firstsample-tagged polypeptides and the second sample-tagged polypeptides.The first sample-tagged polypeptides and the second sample-taggedpolypeptides may be combined prior to contact with the plurality ofmolecular identifier labels. Determining the number of different labeledpolypeptides may comprise detecting at least a portion of the labeledpolypeptide. Detecting at least a portion of the labeled polypeptide maycomprise detecting at least a portion of the sample tag, molecularidentifier label, polypeptide, or a combination thereof. In someembodiments, the detectable tag comprises an L-DNA polynucleotidesequence.

Sequencing

In some embodiments, determining the number of different labeled nucleicacids may comprise determining the sequence of the labeled nucleic acidor any product thereof (e.g., labeled-amplicons, labeled-cDNAmolecules). In some embodiments, an amplified target nucleic acid may besubjected to sequencing. Determining the sequence of the labeled nucleicacid or any product thereof may comprise conducting a sequencingreaction to determine the sequence of at least a portion of the sampletag, molecular identifier label, at least a portion of the labelednucleic acid, a complement thereof, a reverse complement thereof, or anycombination thereof. In some embodiments, only the sample tag or aportion of the sample tag is sequenced. In some embodiments, only themolecular identifier label or a portion of the molecular identifierlabel is sequenced.

Determining the sequence of the labeled nucleic acid or any productthereof may be performed by sequencing methods such as Helioscope(Registered Trademark) single molecule sequencing, Nanopore DNAsequencing, Lynx Therapeutics' Massively Parallel Signature Sequencing(MPSS), 454 pyrosequencing, Single Molecule real time (RNAP) sequencing,Illumina (Solexa) sequencing, SOLiD sequencing, Ion Torrent, Ionsemiconductor sequencing, Single Molecule SMRT (Registered Trademark)sequencing, Polony sequencing, DNA nanoball sequencing, and VisiGenBiotechnologies approach. Alternatively, determining the sequence of thelabeled nucleic acid or any product thereof may use sequencingplatforms, including, but not limited to, Genome Analyzer IN, HiSeq, andMiSeq offered by Illumina, Single Molecule Real Time (SMRT (RegisteredTrademark)) technology, such as the PacBio RS system offered by PacificBiosciences (California) and the Solexa Sequencer, True Single MoleculeSequencing (tSMS (Registered Trademark)) technology such as theHeliScope (Registered Trademark) Sequencer offered by Helicos Inc.(Cambridge, Mass.). In some embodiments, the sequencing reaction canoccur on a solid or semi-solid support, in a gel, in an emulsion, on asurface, on a bead, in a drop, in a continuous follow, in a dilution, orin one or more physically separate volumes.

Sequencing may comprise sequencing at least about 10, 20, 30, 40, 50,60, 70, 80, 90, 100 or more nucleotides or base pairs of the labelednucleic acid. In some embodiments, sequencing comprises sequencing atleast about 200, 300, 400, 500, 600, 700, 800, 900, 1000 or morenucleotides or base pairs of the labeled nucleic acid. In otherembodiments, sequencing comprises sequencing at least about 1500; 2,000;3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or morenucleotides or base pairs of the labeled nucleic acid.

Sequencing may comprise at least about 200, 300, 400, 500, 600, 700,800, 900, 1000 or more sequencing reads per run. In some instances,sequencing comprises sequencing at least about 1500; 2,000; 3,000;4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or more sequencingreads per run. Sequencing may comprise less than or equal to about1,600,000,000 sequencing reads per run. Sequencing may comprise lessthan or equal to about 200,000,000 reads per run.

Cells

The cell as described in the present disclosure may be a cell from ananimal (e.g., human, rat, pig, horse, cow, dog, mouse). In someinstances, the cell is a human cell. The cell may be a fetal human cell.The fetal human cell may be obtained from a mother pregnant with thefetus. The cell may be a cell from a pregnant mother. The cell may be acell from a vertebrate, invertebrate, fungi, archae, or bacteria. Thecell may be from a multicellular tissue (e.g., an organ (e.g., brain,liver, lung, kidney, prostate, ovary, spleen, lymph node, thyroid,pancreas, heart, skeletal muscle, intestine, larynx, esophagus, andstomach), a blastocyst). The cell may be a cell from a cell culture. Thecell may be a HeLa cell, a K562 cell, a Ramos cell, a hybridoma, a stemcell, an undifferentiated cell, a differentiated cell, a circulatingcell, a CHO cell, a 3T3 cell, and the like.

Circulating diseased cells that can be used in the methods of thepresent disclosure include all types of circulating cells that may beaffected by a disease or condition or infected by an infectious agent. Acirculating cell refers to a cell present in the bodily fluid. Acirculating cell may not necessarily circulate throughout the entirebody or in the circulatory system. For example, a circulating cell maybe present locally, such as in synovial fluid, or cerebrospinal fluid,or lymph fluid. A circulating diseased cell may also be detached from atissue or organ that has been affected by a disease or condition orinfected by an infectious agent. In other embodiments, the circulatingdiseased cells can be a mixture of different types of circulatingdiseased cells.

In some embodiments, the cell is a cancerous cell. Non-limiting examplesof cancer cells may include a prostate cancer cell, a breast cancercell, a colon cancer cell, a lung cancer cell, a brain cancer cell, andan ovarian cancer cell. In some embodiments, the cell is from a cancer(e.g., a circulating tumor cell). Non-limiting examples of cancers mayinclude, adenoma, adenocarcinoma, squamous cell carcinoma, basal cellcarcinoma, small cell carcinoma, large cell undifferentiated carcinoma,chondrosarcoma, and fibrosarcoma.

In some embodiments, the cell is a rare cell. A rare cell can be acirculating tumor cell (CTC), circulating epithelial cell (CEC),circulating stem cell (CSC), stem cells, undifferentiated stem cells,cancer stem cells, bone marrow cells, progenitor cells, foam cells,fetal cells, mesenchymal cells, circulating endothelial cells,circulating endometrial cells, trophoblasts, immune system cells (hostor graft), connective tissue cells, bacteria, fungi, or pathogens (forexample, bacterial or protozoa), microparticles, cellular fragments,proteins and nucleic acids, cellular organelles, other cellularcomponents (for example, mitochondria and nuclei), and viruses.

In some embodiments, the cell is from a tumor. In some embodiments, thetumor is benign or malignant. The tumor cell may comprise a metastaticcell. In some embodiments, the cell is from a solid tissue thatcomprises a plurality of different cell types (e.g., differentgenotypes).

Samples

In some embodiments, the sample that includes the template nucleic acid,e.g. DNA and/or RNA, may be combined into the reaction mixture in anamount sufficient for producing a product. In some embodiments, thesample is combined into the reaction mixture such that the finalconcentration of DNA and/or RNA in the reaction mixture is from about 1fg/μL to about 10 μg/μL, from about 1 μg/μL to about 5 μg/μL, from about0.001 μg/μL to about 2.5 μg/μL, from about 0.005 μg/μL to about 1 μg/μL,from about 0.01 μg/μL to about 0.5 μg/μL, from about 0.1 μg/μL to about0.25 μg/μL. In some embodiments, the sample that includes the templateis isolated from a single cell. In some embodiments, the sample thatincludes the template is isolated from about 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, 100, 500 or more cells.

In some embodiments, the template is DNA, an RNA, or a combination ofDNA and RNA. In some embodiments, the template is a fragment or degradedDNA, a fragment or degraded RNA, or a combination of fragment ordegraded DNA and fragment or degraded RNA. In some embodiments, thetotal amount of template is the total amount of template in a sample. Insome embodiments, the total amount of template is the total amount oftemplate in a reaction mixture. In some embodiments, the total amount oftemplate is the total amount of template in one pot or a single vessel.In some embodiments, the total amount of template is the total amount oftemplate in one pot or a single vessel reaction. In some embodiments,the total amount of the template is from about 1 femtomolar (fM) toabout 100 micromolar, from about 0.0001 micromolar to about 0.01micromolar, from about 0.0001 micromolar to about 0.1 micromolar, fromabout 40 femtomolar to about 0.01 micromolar, from about 50 femtomolarto about 500 femtomolar, from about 50 femtomolar to about 0.01micromolar, from about 50 femtomolar to about 0.1 micromolar, from about50 femtomolar to about 500 picomolar, from about 50 femtomolar to about500 nanomolar, from about 50 femtomolar to about 500 micromolar, fromabout 50 femtomolar to about 1 picomolar, from about 40 femtomolar toabout 1 nanomolar, from about 1 femtomolar to about 1 picomolar. In someembodiments, the total amount of template is equal to or at least aboutor lower than about 1000 micromolar, equal to or at least about or lowerthan about 500 micromolar, equal to or at least about or lower thanabout 250 micromolar, equal to or at least about or lower than about 100micromolar, equal to or at least about or lower than about 50micromolar, equal to or at least about or lower than about 25micromolar, equal to or at least about or lower than about 10micromolar, equal to or at least about or lower than about 1 micromolar,equal to or at least about or lower than about 0.1 micromolar, equal toor at least about or lower than about 0.01 micromolar, equal to or atleast about or lower than about 0.001 micromolar, equal to or at leastabout or lower than about 0.0001 micromolar, equal to or at least aboutor lower than about 2000 nanomolar, equal to or at least about or lowerthan about 500 nanomolar, equal to or at least about or lower than about250 nanomolar, equal to or at least about or lower than about 200nanomolar, equal to or at least about or lower than about 50 nanomolar,equal to or at least about or lower than about 25 nanomolar, equal to orat least about or lower than about 20 nanomolar, equal to or at leastabout or lower than about 2 nanomolar, equal to or at least about orlower than about 0.2 nanomolar, equal to or at least about or lower thanabout 0.01 nanomolar, equal to or at least about or lower than about0.001 nanomolar, equal to or at least about or lower than about 0.0001nanomolar, equal to or at least about or lower than about 3000picomolar, equal to or at least about or lower than about 500 picomolar,equal to or at least about or lower than about 250 picomolar, equal toor at least about or lower than about 300 picomolar, equal to or atleast about or lower than about 50 picomolar, equal to or at least aboutor lower than about 25 picomolar, equal to or at least about or lowerthan about 30 picomolar, equal to or at least about or lower than about3 picomolar, equal to or at least about or lower than about 0.3picomolar, equal to or at least about or lower than about 0.01picomolar, equal to or at least about or lower than about 0.001picomolar, equal to or at least about or lower than about 0.0001picomolar, equal to or at least about or lower than about 5000femtomolar, equal to or at least about or lower than about 500femtomolar, equal to or at least about or lower than about 250femtomolar, equal to or at least about or lower than about 50femtomolar, equal to or at least about or lower than about 25femtomolar, equal to or at least about or lower than about 10femtomolar, equal to or at least about or lower than about 1 femtomolar,equal to or at least about or lower than about 0.1 femtomolar, equal toor at least about or lower than about 0.01 femtomolar, equal to or atleast about or lower than about 0.001 femtomolar, equal to or at leastabout or lower than about 0.0001 femtomolar.

In some embodiments, the sample may be obtained from a biological sampleobtained from a subject. In some embodiments, a sample comprisescirculating tumor DNA sample and/or a tissue sample. In someembodiments, the biological sample comprises a cell-free biologicalsample. In some embodiments, the biological sample comprises acirculating tumor DNA sample. In some embodiments, the biological samplecomprises a biopsy sample. In some embodiments, the biological samplecomprises a tissue sample. In some embodiments, the biological samplecomprises liquid biopsy. In some embodiments, the biological samplecomprises cell-free DNA. In some embodiments, the biological sample canbe a solid biological sample, e.g., a tumor sample. In some embodiments,a sample from a subject can comprise at least about 1%, at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or at least about 100% tumor cells or nucleic acidfrom a tumor. The solid biological sample can be processed by fixationin a formalin solution, followed by embedding in paraffin (e.g., a FFPEsample). The solid biological sample can be processed by freezing.Alternatively, the biological sample can be neither fixed nor frozen.The unfixed, unfrozen sample can be stored in a solution configured forthe preservation of nucleic acid. The solid biological sample canoptionally be subjected to homogenization, sonication, French press,dounce, freeze/thaw, which can be followed by centrifugation.

In some embodiments, the sample can be a liquid biological sample. Insome embodiments, the liquid biological sample can be a blood sample(e.g., whole blood, plasma, or serum). A whole blood sample can besubjected to separation of cellular components (e.g., plasma, serum) andcellular components by use of a Ficoll reagent. In some embodiments, theliquid biological sample can be a urine sample. In some embodiments, theliquid biological sample can be a perilymph sample. In some embodiments,the liquid biological sample can be a fecal sample. In some embodiments,the liquid biological sample can be saliva. In some embodiments, theliquid biological sample can be semen. In some embodiments, the liquidbiological sample can be amniotic fluid. In some embodiments, the liquidbiological sample can be cerebrospinal fluid. In some embodiments, theliquid biological sample can be bile. In some embodiments, the liquidbiological sample can be sweat. In some embodiments, the liquidbiological sample can be tears. In some embodiments, the liquidbiological sample can be sputum. In some embodiments, the liquidbiological sample can be synovial fluid. In some embodiments, the liquidbiological sample can be vomit. In some embodiments, the liquidbiological sample can be a cell-free sample. In some specificembodiments, the cell-free sample can be a cell-free plasma sample.

Polynucleotides in a sample (which can be referred to as input nucleicacid or input) can comprise DNA. The input nucleic acid can be complexDNA, such as double-stranded DNA, genomic DNA or mixed nucleic acidsfrom more than one organism. Polynucleotides in the sample can compriseRNA. The RNA can be obtained and purified. RNA can include RNAs inpurified or unpurified form, which include, but are not limited to,mRNAs, tRNAs, snRNAs, rRNAs, retroviruses, small non-coding RNAs,microRNAs, polysomal RNAs, pre-mRNAs, intronic RNA, viral RNA, cell-freeRNA and fragments thereof. The non-coding RNA, or ncRNA may includesnoRNAs, microRNAs, siRNAs, piRNAs and long nc RNAs. Polynucleotides inthe sample can comprise cDNA. The cDNA can be generated from RNA, e.g.,mRNA. The cDNA can be single or double stranded. The input DNA can bemitochondrial DNA. The input DNA can be cell-free DNA. The cell-free DNAcan be obtained from, e.g., a serum or plasma sample. The input DNA canbe from more than one individual or organism. The input DNA can bedouble stranded or single stranded.

In some embodiments, samples can be collected over a period of time.Samples can be collected over regular time intervals, or can becollected intermittently over irregular time intervals. Nucleic acidsfrom different samples can be compared, e.g., to monitor progression orrecurrence of a condition or disease.

In some instances, a sample can be collected by core biopsy. In someembodiments, a sample can be collected as a purified nucleic acid.Examples of such purified samples can include precipitated nucleic acidaffixed to filter paper, phenol-chloroform extractions, nucleic acidpurified by kit purification (e.g. Quigen Miniprep (RegisteredTrademark) and the like), or gel purified nucleic acid as exemplaryexamples.

The sample of the disclosure may be a sample from an animal (e.g.,human, rat, pig, horse, cow, dog, mouse). In some instances, the sampleis a human sample. The sample may be a fetal human sample. The samplemay be from a multicellular tissue (e.g., an organ (e.g., brain, liver,lung, kidney, prostate, ovary, spleen, lymph node, thyroid, pancreas,heart, skeletal muscle, intestine, larynx, esophagus, and stomach), ablastocyst). The sample may be a cell from a cell culture.

The sample may comprise a plurality of cells. The sample may comprise aplurality of the same type of cell. The sample may comprise a pluralityof different types of cells. The sample may comprise a plurality ofcells at the same point in the cell cycle and/or differentiationpathway. The sample may comprise a plurality of cells at differentpoints in the cell cycle and/or differentiation pathway. A sample maycomprise a plurality of samples.

The plurality of samples may comprise at least 5, 10, 20, 30, 40, 50,60, 70, 80, 90 or 100 or more samples. The plurality of samples maycomprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900 or1000 or more samples. The plurality of samples may comprise at leastabout 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 samples, 9000, or10,000 samples, or 100,000 samples, or 1,000,000 or more samples. Theplurality of samples may comprise at least about 10,000 samples.

The one or more nucleic acids in the first sample may be different fromone or more nucleic acids in the second sample. The one or more nucleicacids in the first sample may be different from one or more nucleicacids in a plurality of samples. The one or more nucleic acids maycomprise a length of at least about 1 nucleotide, 2 nucleotides, 5nucleotides, 10 nucleotides, 20 nucleotides, 50 nucleotides, 100nucleotides, 200 nucleotides, 300 nucleotides, 500 nucleotides, 1000nucleotides, 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, 5000nucleotides, 10,000 nucleotides, 100,000 nucleotides, 1,000,000nucleotides.

The first sample may comprise one or more cells and the second samplemay comprise one or more cells. The one or more cells of the firstsample may be of the same cell type as the one or more cells of thesecond sample. The one or more cells of the first sample may be of adifferent cell type as one or more different cells of the plurality ofsamples. The cell type may be chondrocyte, osteoclast, adipocyte,myoblast, stem cell, endothelial cell or smooth muscle cell. The celltype may be an immune cell type. The immune cell type may be a T cell, Bcell, thrombocyte, dendritic cell, neutrophil, macrophage or monocyte.

The plurality of samples may comprise one or more malignant cell. Theone or more malignant cells may be derived from a tumor, sarcoma orleukemia.

The plurality of samples may comprise at least one bodily fluid. Thebodily fluid may comprise blood, urine, lymphatic fluid, saliva. Theplurality of samples may comprise at least one blood sample.

The plurality of samples may comprise at least one cell from one or morebiological tissues. The one or more biological tissues may be a bone,heart, thymus, artery, blood vessel, lung, muscle, stomach, intestine,liver, pancreas, spleen, kidney, gall bladder, thyroid gland, adrenalgland, mammary gland, ovary, prostate gland, testicle, skin, adipose,eye or brain.

The biological tissue may comprise an infected tissue, diseased tissue,malignant tissue, calcified tissue or healthy tissue.

The plurality of samples may be from one or more sources. The pluralityof samples may be from two or more sources. The plurality of samples maybe from one or more subjects. The plurality of samples may be from twoor more subjects. The plurality of samples may be from the same subject.The one or more subjects may be from the same species. The one or moresubjects may be from different species. The one or more subjects may behealthy. The one or more subjects may be affected by a disease, disorderor condition. The plurality of samples may comprise cells of an originselected from a mammal, bacteria, virus, fungus or plant. The one ormore samples may be from a human, horse, cow, chicken, pig, rat, mouse,monkey, rabbit, guinea pig, sheep, goat, dog, cat, bird, fish, frog andfruit fly.

In some embodiments, the plurality of samples may be obtainedconcurrently. The plurality of samples may be obtained at the same time.The plurality of samples may be obtained sequentially. The plurality ofsamples may be obtained over a course of years, 100 years, 10 years, 5years, 4 years, 3 years, 2 years or 1 year of obtaining one or moredifferent samples. One or more samples may be obtained within about oneyear of obtaining one or more different samples. One or more samples maybe obtained within 12 months, 11 months, 10 months, 9 months, 8 months,7 months, 6 months, 4 months, 3 months, 2 months or 1 month of obtainingone or more different samples. One or more samples may be obtainedwithin 30 days, 28 days, 26 days, 24 days, 21 days, 20 days, 18 days, 17days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or one dayof obtaining one or more different samples. One or more samples may beobtained within about 24 hours, 22 hours, 20 hours, 18 hours, 16 hours,14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1hour of obtaining one or more different samples. One or more samples maybe obtained within about 60 sec, 45 sec, 30 sec, 20 sec, 10 sec, 5 sec,2 sec or 1 sec of obtaining one or more different samples. One or moresamples may be obtained within less than one second of obtaining one ormore different samples.

In some embodiments, the present disclosure provides methods, kits andcompositions for diagnosing, monitoring, and/or prognosing a status oroutcome of a disease or condition in a subject. Generally, the methodcomprises (a) labeling one or more molecules (e.g., fragmented ordegraded nucleic acid, DNA or RNA) from one or more samples to produceone or more labeled nucleic acids; (b) amplifying the one or morelabeled nucleic acids (c) detecting and/or quantifying the one or morelabeled nucleic acids; and (d) diagnosing, monitoring, and/or prognosinga status or outcome of a disease or condition in a subject based on thedetecting and/or quantifying of the one or more labeled nucleic acids.In some embodiments, the one or more labeled nucleic acid is indicativeof a disease, e.g. cancer. In some embodiments, the one or more labelednucleic acid is present in a femtomolar range. The method may furthercomprise determining a therapeutic regimen. The one or more of samplesmay comprise one or more samples from a subject suffering from a diseaseor condition. The one or more samples may comprise one or more samplesfrom a healthy subject. The one or more samples may comprise one or moresamples from a control.

Monitoring a disease or condition may further comprise monitoring atherapeutic regimen. Monitoring a therapeutic regimen may comprisedetermining the efficacy of a therapeutic regimen. In some instances,monitoring a therapeutic regimen comprises administrating, terminating,adding, or altering a therapeutic regimen. Altering a therapeuticregimen may comprise increasing or reducing the dosage, dosingfrequency, or mode of administration of a therapeutic regimen. Atherapeutic regimen may comprise one or more therapeutic drugs. Thetherapeutic drugs may be an anticancer drug, antiviral drug,antibacterial drug, antipathogenic drug, or any combination thereof. Insome embodiments, amplification of a target sequence can comprise atleast a part of a genome of an organism. In some embodiments, at leastabout 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%,about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%,about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%,about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%,about 46%, about 47%, about 48%, about 49%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% ofthe genome of an organism can be amplified and/or analyzed.

In some embodiments, amplification of a target sequence can comprise atleast a part of a transcriptome of an organism. In some embodiments, atleast about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%,about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%,about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%,about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about45%, about 46%, about 4′7%, about 48%, about 49%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100% of a transcriptome of an organism can be amplified and analyzed.

In some embodiments, the characteristic that improves enzyme property isselected from the group consisting of increased stability (e.g.,increased thermostability), increased specific activity, increasedprotein expression, increased processivity, increased stranddisplacement, increased end-to-end template jumping, and increasedfidelity.

EXAMPLES

The following specific examples are illustrative and non-limiting. Theexamples described herein reference and provide non-limiting support tothe various embodiments described in the preceding sections.

Example 1: Expression and Purification

Small and medium scale: Expression vector pET-45b caring modified R2non-long terminal repeat (LTR) retrotransposon or a modified R2 reversetranscriptase was transformed into E. coli BL21 (DE3). TABLE 3 belowshows two examples of modified R2 enzyme variants of the presentdisclosure, P2 and P8 variants. For expression, pre-culture was setup in2 ml LB with 100 μM Corbenicillin and grown overnight for about 8 to 12hours at room temperature. After about 8 to 12 h, 200 μL of thepre-culture was transferred to 25 mL of an auto-induction expressionmedia, Overnight Express TB (Novagen), and shaker-incubated at roomtemperature for 36 hours to 48 hours. Cells were harvested bycentrifugation at 8000×g for 10 min at 4-8° C. The biomass-pellet wasfrozen at −20° C. for a minimum of 1 h.

TABLE 3 P2 pET45b(+)-R2- MetAHHHHHHVGTVGTGGGSGGASTALKTAGRRNDLHDDRTASvariant N-terminal AHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSL R2truncation EEMetETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQL enzymeWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMetFNAWMetARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMetPEQFCGYIAHLYDTASTTLAVNNEMetSSPVKVGRGVRQGDPLSPILFNVVMetDLILASLPERVGYRLEMetELVSALAYADDLVLLAGSKVGMetQESISAVDCVGRQMetGLRLNCRKSAVLSMetIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMetLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMetEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMetNWTRFNQMetTSVMetGGGVGStop P8 pET45b(+)-N-MetAHHHHHHVGTVGTGGGSGGASTALKTAGRRNDLHDDRTAS variant terminalAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSL R2 truncation R2EEMetETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQL enzyme KPAWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMetFN (endonucleaseAWMetARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFH mutation)SILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMetPEQFCGYIAHLYDTASTTLAVNNEMetSSPVKVGRGVRQGDPLSPILFNVVMetDLILASLPERVGYRLEMetELVSALAYADDLVLLAGSKVGMetQESISAVDCVGRQMetGLRLNCRKSAVLSMetIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMetLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMetEENKWTVELEPRLRTSVGLRKPAIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMetNWTRFNQMetTSVMetGGGVGStop

Purification: pellet was re-suspended in 0.5 mL lysis buffer (0.5 mLlysis buffer per ⅙ of the biomass) and incubated for 30 minutes at roomtemperature. Lysis buffer composition: 1× BugBuster, 100 mM SodiumPhosphate, 0.1% Tween, 2.5 mM TCEP, 3 μL Protease inhibitor mix (Roche),50 μg lysozyme, 0.5 μL DNaseI (2,000 units/ml, from NEB). Afterincubation, the lysate was mixed with equal volume (0.5 mL) ofHis-binding buffer (50 mM Sodium Phosphate pH 7.7, 1.5M Sodium Chloride,2.5 mM TCEP, 0.1% Tween, 0.03% Triton X-100, and 10 mM Imidazole) andincubated at room temperature for about 10-15 minutes. After incubation,the lysate was centrifuged at 10000×g for about 15 min at a temperaturefrom about 4° C. to about 8° C. Pellet was then mixed with 250 μL ofHis-Affinity Gel (His-Spin Protein Miniprep by Zymo Research) accordingto manufacturer's protocol. After the binding step, the His-Affinity Gelwas washed three times with Washing buffer (50 mM Sodium Phosphate pH7.7, 750 mM Sodium Chloride, 0.1% Tween, 0.03% Triton X-100, 2.5 mMTCEP, and 50 mM Imidazole). The R2 reverse transcriptase (RT) (e.g.,modified enzyme) was eluted with 150 μL of elution buffer (50 mM SodiumPhosphate pH 7.7, 300 mM Sodium Chloride, 2.5 mM TCEP, 0.1% Tween, and250 mM Imidazole) and either used directly or frozen in 30% glycerol.This protocol can be adjusted for expression and purification ofmutagenesis and for screening. For example, a similar protocol can beadjusted to a plate format, such as 2 mL of the Overnight Express TB(Novagen) instead of 25 mL can be used, and the purification step cancomprise 96 well spin plates with nickel-immobilized resin.

Result: After purification, samples were analyzed using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), 4-12%polyacrylamide, Bis-Tris (FIG. 10). SDS-PAGE analysis in FIG. 10illustrates samples containing a full length wild type R2 and truncatedR2 non-LTR retrotransposon. The results showed that no clear product forthe full length wild type R2 was observed, while there was a significantexpression level for the truncated R2 non-LTR retrotransposon. Theeluted sample showed high yield of the truncated R2 non-LTRretrotransposon that was His-tag affinity purified with IMAC. FIG. 10lanes are as follow: M indicates a marker, P indicates a pellet(insoluble), S indicates a supernatant (soluble), and E indicates elutedsamples (His-tag affinity purified R2).

Example 2: Surrogate/Diagnostic Assays

Example of reverse transcriptase (RT) activity assay: Assay can be usedto compare enzyme activity, active fraction, stability (e.g.,thermostability), and robustness of the mutants (e.g., modifiedenzymes). RT activity and active fraction(s) were estimated based onprimer extension assay by comparing fraction(s) of extended tonon-extended DNA primer using various template/primer and enzymeconcentrations. Extension assay was conducted with and without theaddition of a DNA trap. Example protocol: annealed 0.2 μMtemplate/primer with fluorescently labeled primer was pre-incubated withvarious concentrations of R2 RT (relative to template/primer 0.1 to4-fold) at room temperature for 20 minutes. Pre-incubation conditionsincluded 40 mM Tris pH 7.5, 200 mM NaCl, 5 mM TCEP, and 0.1% Tween.Extension started with the addition of MgCl₂ (5 mM, final) and dNTPs (25μM of each, final) and optionally a DNA trap (unlabeled DNA oligo duplexat 3 μM, final, or heparin). The addition of trap DNA helps to estimateRT active fraction(s). The reaction was then incubated for 10 minutesand stopped with EDTA (50 mM, final) or formamide (50%, final). Theproduct of the reaction was analyzed with 15% PAGE-Urea. An example of atemplate sequence used is rCrArG rUrCrA rGrUrC rArGrU rCrArG rUrCrArGrUrG rCrCrA rArArU rGrCrC rUrCrG rUrCrA rUrC and of a primer is/56-FAM/TGATGACGAGGCATTTGGC.

Example of end-to-end template jumping assay: Primer extension assaywith two templates where one template is annealed to a fluorescentlylabeled primer (donor template) and the other is primer-free (acceptornucleic acid). Example protocol: annealed 0.1 μM template/primer withfluorescently labeled primer (alternatively the product of the reactioncan be stained with Syber Gold) was pre-incubated with variousconcentrations of R2 RT (relative to template/primer 0.1 to 4-fold) atroom temperature for 20 minutes. Pre-incubation conditions included 40mM Tris pH 7.5, 200 mM NaCl, 5 mM TCEP, and 0.1% Tween. Extensionstarted with the addition of MgCl₂ (5 mM, final), dNTPs (50 μM of each,final) and the acceptor nucleic acid at various concentrations (rangefrom about 0.01 μM to about 5 μM). The reaction was then incubated for30 min-1 h and stopped with EDTA (50 mM, final) or formamide (50%,final). The product of the reaction was analyzed with 15% PAGE-Urea.Templates: the templates were generated by in vitro RNA synthesis withT7 RNA polymerase based on the DNA template generated in a PCR reactionwith two primers, one of which included a T7 promoter sequence (i.e., afirst primer). The second primer was also used as a DNA primer in thedonor template/primer protocol. The product of the reaction was thenanalyzed with 15% PAGE-Urea. Example of materials used: template for PCRamplification pUC18 with T7 primerCTGCAGTAATACGACTCACTATAGGATCCTCTAGAGTCGACCTGC; donor primerGCCATTCGCCATTCAGGCTGC (used for both PCR amplification and priming atthe donor RNA template); RNA template (˜190 nucleotides); acceptornucleic acid—G-block PCR templateACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCC; two primers for PCR amplification (a T7 primerACGGCCAGTGAATTGTAATACGAC and a second primer GGAAACAGCTATGACCATG).

Example of processivity assay: processivity assay was analyzed based onprimer extension and product formation using a15% PAGE-Urea, or a 1.2%agarose gel, or a 2% agarose gel. Product length distribution wasanalyzed with densitometry. Example protocol: annealed 0.05-0.1 μMtemplate/primer with fluorescently labeled primer (alternatively productof the reaction can be stained with Syber Gold) was pre-incubated withvarious concentration of R2 RT (0.1 to 4-fold relative totemplate/primer) for 20 minutes at room temperature. Pre-incubationconditions: 40 mM Tris pH 7.5, 200 mM NaCl, 5 mM TCEP, and 0.1% Tween.Extension started with addition of MgCl₂ (5 mM, final), dNTPs (50 μM ofeach, final), and optionally a DNA trap (unlabeled DNA oligo duplex at 3μM, final). The reaction was then incubated for 30 min-1 h and stoppedwith EDTA (50 mM, final) or formamide (50%, final). Templates: thetemplates were generated by in vitro RNA synthesis with T7 RNApolymerase based on the DNA template generated in a PCR reaction withtwo primers, one of which included a T7 promoter sequence. The secondprimer was also used as a DNA primer in the donor template/primerprotocol. The product of the reaction was analyzed with a 15% PAGE-Urea,or a 1.2% agarose gel, or a 2% agarose gel. Materials included: templatefor PCR amplification pUC18 with T7 primerCTGCAGTAATACGACTCACTATAGGATCCTCTAGAGTCGACCTGC, RT primerCAGGGTTATTGTCTCATGAGCG (used for both PCR amplification and priming atthe donor RNA template), and RNA template (˜600 nucleotides).

Example of Random priming: Longer RNA template(s) with several primerswith adapters or random primers with adapters; product analysis isperformed after PCR amplification to compare product's lengthdistribution (one primer is specific to the 5′-end of the template andthe second primer is complementary to the adapter sequence).

Example 3: Activity and Template Jumping Experiment Using Synthetic RNA

Modified R2 enzyme showed activity and template jumping properties (FIG.11). Experiment described in TABLE 4 below, and represented by FIG. 11,showed that there was no activity when the reaction lacked an enzyme(lane 2), that there was extension product but no template jumping whenthe reaction lacked an acceptor nucleic acid (lane 3), and that templatejumping was dependent on the concentration of the acceptor nucleic acid(lanes 4 and 5). Lane 6 shows that although the enzyme MMLV is capableof product extension, no apparent product was observed (TABLE 4 and FIG.11). In brief, the reactions contained 0.25 mM of dNTPs, R2 buffer orMMLV buffer, 0.4 μM template/primer, acceptor nucleic acid (0 to 1 μM),modified R2 enzyme (0 to 0.023 μg/μl) or MMLV (2 U/μl), and H₂O. Thereactions containing the R2 enzyme or the R2 buffer were incubated at30° C. for 1 hour (lanes 2-5). The reaction containing the MMLV enzymewas incubated at 42° C. for 1 hour (lane 6). Products were analyzedusing 15% PAGE-Urea gel.

TABLE 4 Reaction Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 H₂O 36.5 μl 33 μl32.75 μl 36.25 μl 34 μl 5X R2 buffer 10 μl 10 μl 10 μl 10 μl 0 μl 5XMMLV buffer 0 μl 0 μl 0 μl 0 μl 10 μl 10 mM dNTPs 1.25 μl 1.25 μl 1.25μl 1.25 μl 1.25 μl 10 μM template/primer 2 μl 2 μl 2 μl 2 μl 2 μl(P173 + P174) 100 μM P181 TSO 0.25 μl 0 μl 0.25 μl 0.5 μl 0.25 μl(acceptor nucleic acid) 0.3 μg/μl P8 (R2 enzyme) 0 μl 3.75 μl 3.75 μl3.75 μl 0 μl 40 unit/μl MMLV-RT 0 μl 0 μl 0 μl 0 μl 2.5 μl (Clontech)Total 50 μl 50 μl 50 μl 50 μl 50 μl

Sequences:

P173 (RNA template) CAGUCAGUCAGUCAGUCAGUGCCAAAUGCCUCGUCAUCP174 (fluorescently labeled /56-FAM/TGATGACGAGGCATTTGGC primer)P181 (acceptor nucleic acid) GTTAATAACGAAATGAGCAGCCrGrGrG

Example 4: 1-Pot (Single Vessel) Reaction

This experiment was designed to demonstrate a method for preparing 1-pot(single vessel) RNA library (TABLE 5). This experiment successfullycaptured a 200 bp RNA molecule in 1-pot (single vessel) reaction. Aschematic of the workflow is shown in FIG. 3 and FIG. 12A. The templateused in this experiment was a 200 base pair synthetic RNA (donortemplate) generated using T7 in vitro transcription protocol. Thecontrols included: no enzyme control, no acceptor control, no acceptorand no donor template with primer control, and MMLV (TABLE 5). Othercontrols used in this experiment included using only one primer duringPCR amplification (FIG. 12B, numbers 5 and 6).

In brief, the R2 enzyme reaction (RT reaction) included H₂O, R2 buffer,0.25 mM of dNTPs, 0.025 μM 200 bp RNA/primer (donor template withprimer), acceptor nucleic acid (0.15 μM), and 0.023 μg/μl of R2 enzyme(e.g., modified reverse transcriptase enzyme) (TABLE 5, FIG. 12C lane4). Refer to TABLE 5 for the specifics of the other reactions. The R2reaction was then incubated at 30° C. for 1 hour and the MMLV reactionwas incubated at 42° C. for 1 hour. The reactions were then supplementedwith PCR reagents including amplification primers and a hot-startpolymerase (TABLE 6). In brief, 2.5 μl of the RT reaction (i.e., 2.5 μlof the 50 μl reaction from TABLE 5) was supplemented with componentsnecessary for PCR amplification. The PCR amplification reaction for R2enzyme included H₂O, SYBR FAST master mix, 0.5 μM each of P186 primerand P170 primer, and template (e.g., 2.5 μl of the RT reaction). The PCRcondition was 95° C. for 3 minutes and 30 cycles of 95° C. for 3 secondsand 62° C. for 20 seconds. The reactions were then increased to 68° C.No purification step was needed during this experiment (e.g., the RTreaction was not purified prior to PCR). In some instances, the initialRT reaction (or R2 reaction) was diluted 10 fold during the PCR step,e.g., 2.5 μl RT reaction in 25 μl total volume (see, TABLE 6). In someinstances, 10 μl RT reaction and 90 μl PCR reagents can be added to thesame tube (hence a 1-pot or single vessel reaction). The gel in FIG. 12Bshows that the right size amplicon was generated using the R2 enzymereaction (lane 4). The other lanes did not show amplicon formation (lane1: ladder; lane 2: no enzyme control; lane 3: MMLV control; lane 5: noacceptor control; lane 6: no donor control; and lane 7: ladder).

TABLE 5 No acceptor control/ no No Enzyme No donor No acceptor donortemplate Reaction control control control with primer R2 enzyme MMLV H₂O32.5 μl 30 μl 33.75 μl 34.375 μl 28.75 μl 36.75 μl 5X R2 buffer 10 μl 10μl 10 μl 10 μl 10 μl 0 μl 5X MMLV buffer 0 μl 0 μl 0 μl 0 μl 0 μl 10 μl10 mM dNTPs 1.25 μl 1.25 μl 1.25 μl 1.25 μl 1.25 μl 1.25 μl 12 μM 200bpRNA 0 μl 0 μl 0 μl 0.625 μl 0 μl 0 μl (donor template without primer)1 μM 1.25 μl 0 μl 1.25 μl 0 μl 1.25 μl 1.25 μl 200 bpRNA/primer (RNA +P187) (donor template with primer) 1 μM P173 5 μl 5 μl 0 μl 0 μl 5 μl 0μl 0.3 μg/μl P2 0 μl 3.75 μl 3.75 μl 3.75 μl 3.75 μl 0 μl (R2 enzyme) 40unit/μl MMLV- 0 μl 0 μl 0 μl 0 μl 0 μl 1.25 μl RT (Clontech) Total 50 μl50 μl 50 μl 50 μl 50 μl 50 μl

TABLE 6 Reagents volume H₂O 7.5 μl 2× SYBRFAST master mix 12.5 μl 10 μMprimer (P186) 1.25 μl 10 μM primer (P170) 1.25 μl 10× template (or RTreaction) 2.5 μl Total 25 μl Thermocycling 95° C. 3 minutes 95° C. 3seconds {close oversize brace} 30 cycles 62° C. 20 seconds 68° C. 5′

Sequences:

P173 (RNA template) CAGUCAGUCAGUCAGUCAGUGCCAAAUGCCUCGUCAUC 200 bp RNAGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCUUGGCACUGGCCGUCGUUUUCAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAACUUAAUCGCCUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGCCCGCACCGAUCGCCCUUCCCAACAGUUGCGCAGCCUGAAUGGCGAAUGGC P186 (amplification primer)CAGTCAGTCAGTCAGTCAGTGCCA P187 (primer) CACGACGTTGTAAAACGACGGCP170 (amplification primer) GCCATTCGCCATTCAGGCTGC

Example 5: 1-Pot (Single Vessel) RNA Library Prep Using Various TemplateAmounts

This experiment was designed to demonstrate a method for preparing 1-pot(single vessel) RNA library using different amounts of template RNA(TABLE 7). A schematic of the workflow is shown in FIG. 3. The templateused in this experiment was a 600 base pair synthetic RNA (donortemplate). The RNA template amounts used in this experiment varied from0 μM, 0.00025 μM, 0.0025 μM, and 0.025 μM. In brief, the reactionsincluded H₂O, R2 buffer or MMLV buffer, 0.25 mM dNTPs, 600 bp RNAtemplate with primer (0 μM, 0.00025 μM, 0.0025 μM, and 0.025 μM),acceptor nucleic acid molecule (6 μM of P181 TSO for MMLV reactions) or0.1 μM of P173 for R2 reactions, and enzyme (0.023 μg/μl of modified R2enzyme or 0.4 U/μl of MMLV). The R2 reactions were incubated at 30° C.for 1 hour (TABLE 7, numbers 1-4) and the MMLV reactions were incubatedat 42° C. for 1 hour (TABLE 7, numbers 5-8). The reactions were thensupplemented with PCR reagents including amplification primers andhot-start polymerase. The PCR conditions are shown in TABLE 8 for the R2reactions (reactions 1-4) and in TABLE 9 for the MMLV reactions(reactions 5-8). The R2 reactions included H₂O, SYBR FAST master mix,0.5 μM each of P186 primer and P172 primer, and 1× template (2.5 μl ofthe RT reaction). The MMLV reactions included H₂O, SYBR FAST master mix,0.5 μM each of P161 primer and P172 primer, and 1× template (2.5 μl ofthe RT reaction). In brief, 2.5 μl of the RT reaction (e.g., withoutcleanup) was incubated with PCR components including SYBRFAST mastermix, primers, and H₂O in a total volume of 25 μl (one pot/tube/singlevessel reaction). The PCR conditions for the R2 reactions were 95° C.for 3 minutes and 30 cycles of 95° C. for 3 seconds, 54° C. for 10seconds, and 64° C. for 20 seconds. The reactions were then increased to68° C. The PCR conditions for the MMLV reactions were 95° C. for 3minutes and 30 cycles of 95° C. for 3 seconds and 64° C. for 20 seconds.The reactions were then increased to 68° C. No purification step wasneeded during the course of this experiment. In some instances, theinitial reaction can be diluted 10-fold during PCR by adding 10 μl RTreaction and 90 μl PCR reagents to the same tube, making it a 1-pot(single vessel) reaction. The results using real-time PCR showed productconversion at various amounts of 600 base pair RNA and showed that theR2 enzyme presented superior conversion efficiency compared to MMLV(FIG. 13). This experiment indicated that even at low amounts oftemplate (0.00025 μM), the R2 enzyme was capable of RNA library prep(FIG. 13, number 4). This is representative of single cell applications.This experiment also showed a 10 fold greater conversion efficiency ascompared to currently available methods.

TABLE 7 Reaction 1 2 3 4 5 6 7 8 H₂O 34.95 μl 34.45 μl 34.9 μl 34.945 μl35.25 μl 34.75 μl 35.2 μl 35.245 μl 5X R2 buffer 10 μl 10 μl 10 μl 10 μl0 μl 0 μl 0 μl 0 μl 5X MMLV 0 μl 0 μl 0 μl 0 μl 10 μl 10 μl 10 μl 10 μlbuffer 10 mM dNTPs 1.25 μl 1.25 μl 1.25 μl 1.25 μl 1.25 μl 1.25 μl 1.25μl 1.25 μl 2.5 μM 0 μl 0.5 μl 0.05 μl 0.005 μl 0 μl 0.5 μl 0.05 μl 0.005μl 600 bpRNA- P172 100 μM P181 0 μl 0 μl 0 μl 0 μl 3 μl 3 μl 3 μl 3 μlTSO 100 μM P173 0.05 μl 0.05 μl 0.05 μl 0.05 μl 0 μl 0 μl 0 μl 0 μl 0.3μg/μl P8 3.75 μl 3.75 μl 3.75 μl 3.75 μl 0 μl 0 μl 0 μl 0 μl (R2 enzyme)40 unit/μl 0 μl 0 μl 0 μl 0 μl 0.5 μl 0.5 μl 0.5 μl 0.5 μl MMLV-RT(Clontech) Total 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl

TABLE 8 Reagents volume H₂O 9.75 μl 2× SYBRFAST master mix 12.5 μl 100μM primer (P186) 0.125 μl 100 μM primer (P172) 0.125 μl 10× template (orRT reaction) 2.5 μl Total 25 μl Thermocycling 95° C. 3 minutes 95° C. 3seconds 54° C. 10 seconds {close oversize brace} 30 cycles 64° C. 20seconds 68° C. 2′

TABLE 9 Reagents volume H₂O 9.75 μl 2× SYBRFAST master mix 12.5 μl 100μM primer (P161) 0.125 μl 100 μM primer (P172) 0.125 μl 10× template (orRT reaction) 2.5 μl Total 25 μl Thermocycling 95° C. 3 minutes 95° C. 3seconds {close oversize brace} 30 cycles 64° C. 20 seconds 68° C. 2′

Sequences:

P173 CAGUCAGUCAGUCAGUCAGUGCCAAAUGCCUCGUCAUC (RNA template) 600 bp RNAGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCUUGGCACUGGCCGUCGUUUUACAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAACUUAAUCGCCUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGCCCGCACCGAUCGCCCUUCCCAACAGUUGCGCAGCCUGAAUGGCGAAUGGCGCCUGAUGCGGUAUUUUCUCCUUACGCAUCUGUGCGGUAUUUCACACCGCAUAUGGUGCACUCUCAGUACAAUCUGCUCUGAUGCCGCAUAGUUAAGCCAGCCCCGACACCCGCCAACACCCGCUGACGCGCCCUGACGGGCUUGUCUGCUCCCGGCAUCCGCUUACAGACAAGCUGUGACCGUCUCCGGGAGCUGCAUGUGUCAGAGGUUUUCACCGUCAUCACCGAAACGCGCGAGACGAAAGGGCCUCGUGAUACGCCUAUUUUUAUAGGUUAAUGUCAUGAUAAUAAUGGUUUCUUAGACGUCAGGUGGCACUUUUCGGGGAAAUGUGCGCGGAACCCCUAUUUGUUUAUUUUUCUAAAUACAUUCAAAUAUGUAUCCGCUCAUGAGACAAUAA CCCUG P186CAGTCAGTCAGTCAGTCAGTGCCA P172 CAGGGTTATTGTCTCATGAGCG P161GTTAATAACGAAATGAGCAGCC P181 GTTAATAACGAAATGAGCAGCCrGrGrG P170GCCATTCGCCATTCAGGCTGC

Example 6: 1-Pot (Single Vessel) RNA Library Prep Using Various TemplateLengths

This experiment was designed to demonstrate a method for preparing 1-pot(single vessel) RNA library using different lengths of template RNA (200bp, 600 bp, and 2000 bp). A schematic of the workflow is shown in FIG.3. In brief, the reactions included H₂O, R2 buffer, 0.25 mM dNTPs, RNAwith primer (e.g., 0.3 μM (200 bp), 0.125 μM (600 bp), or 0.03 μM (2000bp)), 0.1 μM of P173, and enzyme (e.g., 0.023 μg/μl of modified R2enzyme; P2 variant) (TABLE 10). The reactions were incubated at 30° C.for 1 hour. The reactions were then supplemented with PCR reagentsincluding amplification primers and hot-start polymerase. The PCRamplification reactions included H₂O, SYBR FAST master mix, 0.5 μM eachof forward primer and reverse primer, and 2.5 μl of 10× template (RTreaction) (TABLE 11). The forward and reverse primers for the notemplate control reaction were P186 and P183, respectively. The forwardand reverse primers for the 200 bp RNA template reaction were P186 andP170, respectively. The forward and reverse primers for the 600 bp RNAtemplate reaction were P186 and P172, respectively. The forward andreverse primers for the 2000 bp RNA template reaction were P186 andP183, respectively. The PCR conditions for the reactions were 95° C. for3 minutes and 30 cycles of 95° C. for 3 seconds and 62° C. for 60seconds. The reactions were then increased to 68° C. No purificationstep was needed during the course of this experiment. The initialreaction can be diluted 10-fold during PCR by adding 10 μl of the RTreaction and 90 μl PCR reagents to the same tube, making it a 1-pot(single vessel) reaction. The results using real-time PCR showed productconversion at various lengths of RNA template (FIG. 14). This experimentalso illustrates RNA length compatibility. The experiment showscompatibility with several transcript lengths and enables applicationssuch as Iso-seq, VDJ, and others.

TABLE 10 Reaction 1 2 3 4 H₂O 30 μl 27.5 μl 27.5 μl 27.5 μl 5X R2 buffer10 μl 10 μl 10 μl 10 μl 10 mM dNTPs 1.25 μl 1.25 μl 1.25 μl 1.25 μl 2.5μM 600 bpRNA- 0 μl 0 μl 2.5 μl 0 μl P172 0.6 μM 2000 bpRNA- 0 μl 0 μl 0μl 2.5 μl P184 6 μM 200 bpRNA-P170 0 μl 2.5 μl 0 μl 0 μl 1 μM P173 5 μl5 μl 5 μl 5 μl 0.3 μg/μl P2 (R2 3.75 μl 3.75 μl 3.75 μl 3.75 μl enzyme)Total 50 μl 50 μl 50 μl 50 μl

TABLE 11 Reagents volume H₂O 7.5 μl 2× SYBRFAST master mix 12.5 μl 10 μMforward primer 1.25 μl 10 μM reverse primer 1.25 μl 10× template (RTreaction) 2.5 μl Total 25 μl Thermocycling 95° C. 3 minutes 95° C. 3seconds {close oversize brace} 30 cycles 62° C. 60 seconds 68° C. 5′

Sequences:

200 bp RNA GGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCUUGGCACUGGCCGUCGUUUUACAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAACUUAAUCGCCUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGCCCGCACCGAUCGCCCUUCCCAACAGUUGCGCAGCCU GAAUGGCGAAUGGC 600 bp RNAGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCUUGGCACUGGCCGUCGUUUUACAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAACUUAAUCGCCUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGCCCGCACCGAUCGCCCUUCCCAACAGUUGCGCAGCCUGAAUGGCGAAUGGCGCCUGAUGCGGUAUUUUCUCCUUACGCAUCUGUGCGGUAUUUCACACCGCAUAUGGUGCACUCUCAGUACAAUCUGCUCUGAUGCCGCAUAGUUAAGCCAGCCCCGACACCCGCCAACACCCGCUGACGCGCCCUGACGGGCUUGUCUGCUCCCGGCAUCCGCUUACAGACAAGCUGUGACCGUCUCCGGGAGCUGCAUGUGUCAGAGGUUUUCACCGUCAUCACCGAAACGCGCGAGACGAAAGGGCCUCGUGAUACGCCUAUUUUUAUAGGUUAAUGUCAUGAUAAUAAUGGUUUCUUAGACGUCAGGUGGCACUUUUCGGGGAAAUGUGCGCGGAACCCCUAUUUGUUUAUUUUUCUAAAUACAUUCAAAUAUGUAUCCGCUCAUGAGACA AUAACCCUG 2000 bp RNAGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCUUGGCACUGGCCGUCGUUUUACAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAACUUAAUCGCCUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGCCCGCACCGAUCGCCCUUCCCAACAGUUGCGCAGCCUGAAUGGCGAAUGGCGCCUGAUGCGGUAUUUUCUCCUUACGCAUCUGUGCGGUAUUUCACACCGCAUAUGGUGCACUCUCAGUACAAUCUGCUCUGAUGCCGCAUAGUUAAGCCAGCCCCGACACCCGCCAACACCCGCUGACGCGCCCUGACGGGCUUGUCUGCUCCCGGCAUCCGCUUACAGACAAGCUGUGACCGUCUCCGGGAGCUGCAUGUGUCAGAGGUUUUCACCGUCAUCACCGAAACGCGCGAGACGAAAGGGCCUCGUGAUACGCCUAUUUUUAUAGGUUAAUGUCAUGAUAAUAAUGGUUUCUUAGACGUCAGGUGGCACUUUUCGGGGAAAUGUGCGCGGAACCCCUAUUUGUUUAUUUUUCUAAAUACAUUCAAAUAUGUAUCCGCUCAUGAGACAAUAACCCUGAUAAAUGCUUCAAUAAUAUUGAAAAAGGAAGAGUAUGAGUAUUCAACAUUUCCGUGUCGCCCUUAUUCCCUUUUUUGCGGCAUUUUGCCUUCCUGUUUUUGCUCACCCAGAAACGCUGGUGAAAGUAAAAGAUGCUGAAGAUCAGUUGGGUGCACGAGUGGGUUACAUCGAACUGGAUCUCAACAGCGGUAAGAUCCUUGAGAGUUUUCGCCCCGAAGAACGUUUUCCAAUGAUGAGCACUUUUAAAGUUCUGCUAUGUGGCGCGGUAUUAUCCCGUAUUGACGCCGGGCAAGAGCAACUCGGUCGCCGCAUACACUAUUCUCAGAAUGACUUGGUUGAGUACUCACCAGUCACAGAAAAGCAUCUUACGGAUGGCAUGACAGUAAGAGAAUUAUGCAGUGCUGCCAUAACCAUGAGUGAUAACACUGCGGCCAACUUACUUCUGACAACGAUCGGAGGACCGAAGGAGCUAACCGCUUUUUUGCACAACAUGGGGGAUCAUGUAACUCGCCUUGAUCGUUGGGAACCGGAGCUGAAUGAAGCCAUACCAAACGACGAGCGUGACACCACGAUGCCUGUAGCAAUGGCAACAACGUUGCGCAAACUAUUAACUGGCGAACUACUUACUCUAGCUUCCCGGCAACAAUUAAUAGACUGGAUGGAGGCGGAUAAAGUUGCAGGACCACUUCUGCGCUCGGCCCUUCCGGCUGGCUGGUUUAUUGCUGAUAAAUCUGGAGCCGGUGAGCGUGGGUCUCGCGGUAUCAUUGCAGCACUGGGGCCAGAUGGUAAGCCCUCCCGUAUCGUAGUUAUCUACACGACGGGGAGUCAGGCAACUAUGGAUGAACGAAAUAGACAGAUCGCUGAGAUAGGUGCCUCACUGAUUAAGCAUUGGUAACUGUCAGACCAAGUUUACUCAUAUAUACUUUAGAUUGAUUUAAAACUUCAUUUUUAAUUUAAAAGGAUCUAGGUGAAGAUCCUUUUUGAUAAUCUCAUGACCAAAAUCCCUUAACGUGAGUUUUCGUUCCACUGAGCGUCAGACCCCGUAGAAAAGAUCAAAGGAUCUUCUUGAGAUCCUUUUUUUCUGCGCGUAAUCUGCUGCUUGCAAACAAAAAAACCACCGCUACCAGCGGUGGUUUGUUUGCCGGAUCAAGAGCUACCAACUCUUUUUCCGAAGGUAACUGGCUUCAGCAGAGCGCAGAUACCAAAUACUGUCCUUCUAGUGUAGCCGUAGUUAGGCCACCACUUCAAGAACUCUGUAGCACCGCCUACAUACCUCGCUCUGCUAAUCCUGUUACCAGUGGCUGCUGCCAGUGGCGAUAAGUCGUGUCUUACCGGGUUGGACUCA AGACGAUAGUU P186CAGTCAGTCAGTCAGTCAGTGCCA P172 CAGGGTTATTGTCTCATGAGCG P173 (RNACAGUCAGUCAGUCAGUCAGUGCCAAAUGCCUCGUCAUC template) P183TCGTCTTGAGTCCAACCCGGT P170 GCCATTCGCCATTCAGGCTGC P184GAACACAGCATTAGCAGCTCGTCTTGAGTCCAACCCGGT

Example 7: Optimization of NaCl Concentration for Enzyme Activity andTemplate Lumping

This experiment was designed to optimize enzyme activity and templatejumping based on NaCl concentration. In brief, the reactions includedH₂O, 10×R2 buffer, 0.1 mM dNTPs, 0.2 μM RNA template with primer (P173and P174), NaCl (100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, or 800mM), and enzyme (e.g., 0 μg/μl or 0.023 μg/μl of modified R2 enzyme (P2variant R2 enzyme)). The 10×R2 buffer included H₂O, 500 mM Tris-HCl pH7.5, 0.5% tween, and 25 mM DTT (42.5 μl H₂O, 50 μl of 1000 mM Tris-HClpH 7.5, 5 μl of 10% Tween, 2.5 μl of 1000 mM DTT). The reactions wereincubated at room temperature for about 2 hours. The reactions were thensupplemented with 0.5 μM of template (P173) and 5 mM MgCl₂ and incubatedat room temperature for about 1 hour followed by 80° C. for about 5minutes. The reactions were then run on a gel (FIG. 15). Resultssuggested that 300 mM NaCl was optimum for enzyme activity and templatejumping in this experiment (FIG. 15).

TABLE 12 Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Noenzyme NaCl NaCl NaCl NaCl NaCl NaCl NaCl Reaction control 100 mM 200 mM300 mM 400 mM 500 mM 600 mM 800 mM H₂O 16.1 μl 14.6 μl 14.2 μl 13.8 μl13.4 μl 13 μl 12.6 μl 11.8 μl 10X R2 buffer 2 μl 2 μl 2 μl 2 μl 2 μl 2μl 2 μl 2 μl (no MgCl₂; low NaCl) 5000 mM NaCl 0.4 μl 0.4 μl 0.8 μl 1.2μl 1.6 μl 2 μl 2.4 μl 3.2 μl 10 mM dNTPs 0.2 μl 0.2 μl 0.2 μl 0.2 μl 0.2μl 0.2 μl 0.2 μl 0.2 μl 20 μM 0.2 μl 0.2 μl 0.2 μl 0.2 μl 0.2 μl 0.2 μl0.2 μl 0.2 μl template/primer (P173 + P174) 0.3 μg/μl P2 0 μl 1.5 μl 1.5μl 1.5 μl 1.5 μl 1.5 μl 1.5 μl 1.5 μl (R2 enzyme) RT for 2 hours 100 μMP173 0.1 μl 0.1 μl 0.1 μl 0.1 μl 0.1 μl 0.1 μl 0.1 μl 0.1 μl 100 mMMgCl₂ 1 μl 1 μl 1 μl 1 μl 1 μl 1 μl 1 μl 1 μl Total 20 μl 20 μl 20 μl 20μl 20 μl 20 μl 20 μl 20 μl RT for 1 hour then 80° C. for 5 minutes

Example 8: Two Step Purification of R2 Enzyme (P2 Variant) YieldedHigher Activity and Template Lumping

This experiment was designed to optimize the activity and templatejumping properties of an R2 enzyme based on different purificationmethods. In brief, the reactions included H₂O, 10×R2 buffer, 0.1 mMdNTPs, 0.2 μM RNA with primer (P173 and P174), 5 mM MgCl₂, NaCl, 0.5 μMof P173, and enzyme (0.023 μg/μl or 0.045 μg/μl of P2 variant R2 enzymefor one step purification (nickel or heparin) or 0.0075 μg/μl, 0.023μg/μl, or 0.048 μg/μl of P2 variant R2 enzyme for two step purification(nickel and heparin)). The reactions were incubated at room temperaturefor about 2 hours followed by 80° C. for about 5 minutes. The reactionswere then run on a gel (FIGS. 16A and 16B). Results suggested thatreactions where the enzyme was subjected to Ni-NTA nickel affinitycolumn followed by further purification with heparin sepharose affinitycolumn showed higher activity and better template jumping capabilitiesthan reactions subjected to only one purification step (nickel alone orheparin alone) (FIGS. 16A and 16B).

Example 9: DNA Template Allows for R2 Enzyme (P2 Variant) Activity andTemplate Lumping

This experiment demonstrated that a modified reverse transcriptaseenzyme showed activity and template jumping capabilities in the presenceof DNA template (FIG. 17). In short, the reaction included H₂O, 5×R2buffer, 0.25 mM dNTPs, 0.4 μM DNA template with primer (P188 and P189),0 μM or 1.5 μM of DNA template (P188) (FIG. 17, lanes 1 and 2,respectively), and 0 μg/μl or 0.023 μg/μl enzyme (P2 variant R2 enzyme)(FIG. 17, lanes 1 and 2, respectively). The reactions were incubated at30° C. for about 1 hour followed by 80° C. for about 10 minutes. Thereactions were then run on a gel (FIG. 17). Sequences: DNA template P188(AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC GCTCTTCCGATCT); primerP189 (AGATCGGAAGAGCGTCGTGTAG).

Example 10: DNA Fragments can be Captured and Tagged with R2 Enzyme

This experiment showed that a 200 bp DNA fragment (typical size forcfDNA) was captured and tagged in a 1-pot (single vessel) reaction usingthe methods of the present disclosure. Some facts of this experiment: noprior knowledge of the sequence was required and the data provided bythis experiment met the sensitivity requirement (a typical liquid biopsysample has between about 10-30 ng of DNA, a required sensitivity of 0.1%(˜10-30 pg)).

This experiment showed that 1-pot (single vessel) reaction containingDNA fragments (200 bp PCR product prepared by heat denaturation andquick cooling of PCR product) was captured and tagged using an R2 enzyme(P8 variant R2 enzyme). In brief, this experiment included twoapproaches: 1) capture of DNA fragment with RNA priming (schematic shownin FIGS. 5A and 5B); and 2) capture of DNA fragment using RNA donor(schematic shown in FIGS. 6A and 6B). Briefly, the reactions per thefirst approach (RNA priming) included H₂O, 5×R2 buffer, 0.25 mM dNTPs,200 bp DNA fragment (0 ng (no DNA template control (NTC)), 160 pg, 32pg, or 6 pg of DNA template), enzyme (e.g., 0.023 μg/μl P8 variant R2enzyme), and 0.5 μM of P173. The reactions per the second approach (RNAdonor) included H₂O, 5×R2 buffer, 0.25 mM dNTPs, 200 bp DNA fragment (0ng (no DNA template control (NTC)), 160 pg, 32 pg, or 6 pg), enzyme(e.g., 0.023 μg/μl P8 variant R2 enzyme), and 0.2 μM RNA donor(P173+P174). The reactions were then incubated at 30° C. for about 1hour. The reactions were then diluted 1:10 and supplemented with PCRreagents including amplification primers and hot-start polymerase. ThePCR amplification reactions for the first approach (RNA priming)included H₂O, 1×taq master mix with 1×SYBR Green, 0.5 μM of P169, 0.5 μMof P186, and 1× template (10 μl RT reaction in 100 μl total volume forPCR). The PCR amplification reactions for the second approach (RNAdonor) included H₂O, 1×Taq Mastermix with 1×sybr green, 0.5 μM of P169,0.5 μM of P186, and 1× template (10 μl RT reaction in 100 μl totalvolume for PCR). The PCR conditions for the reactions were 95° C. for 3minutes and 30 cycles of 95° C. for 3 seconds, 54° C. for 10 seconds,and 64° C. for 10 seconds. The reactions were then increased to 68° C.for 2′. The length of the PCR products was confirmed on an acrylamidegel. The results showed that the DNA fragment (˜200 bp) was capturedusing either the RNA priming or the donor RNA mechanism without priorknowledge of the DNA sequence. The expected PCR product was about 240bp, which is about the size of the DNA template in addition to the RNAdonor (FIG. 18B). See, FIGS. 18A, 18B, 18C, and 18D.

Sequences:

200 bp DNA fragment CTGCAGTAATACGACTCACTATAGGATCCTCTAGAGTCGACCTGCAGG(PCR product) CATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGC P169CTGCAGTAATACGACTCACTATAGGATCCTCTAGAGTCGACCTGC P186CAGTCAGTCAGTCAGTCAGTGCCA P173 (RNA template)CAGUCAGUCAGUCAGUCAGUGCCAAAUGCCUCGUCAUC P174 TGATGACGAGGCATTTGGC

Example 11: Limit of Detection for DNA Template Using R2 Enzyme

This experiment analyzed the limit of detection for DNA template usingRNA donor strategy for capturing 58 bp DNA oligonucleotide (P188). Inshort, the reactions included H₂O, 5×R2 buffer, 0.25 mM dNTPs, DNAfragment/P188 (0 μM, 0.5 μM, 0.05 μM, 0.005 μM, 0.0005 μM, 0.00005 μM,0.000005 μM, and 0.0000005 μM), enzyme (e.g., 0.023 μg/μl P8 variant R2enzyme), and 0.4 μM RNA with annealed DNA primer (P173/P174). Thereactions were then incubated at 30° C. for about 1 hour and 80° C. forabout 10 minutes. The reactions were then subjected to 5 fold dilutionand supplemented with PCR reagents including amplification primers andhot-start polymerase. The PCR amplification reactions included H₂O,SYBRFASTmaster mix, 0.25 μM of P188, 0.25 μM of P174, and 1× template(2.5 μl of RT reaction in 25 μl total volume). The PCR conditions forthe reactions were 95° C. for 3 minutes and 30 cycles of 95° C. for 3seconds, 54° C. for 10 seconds, and 64° C. for 10 seconds. The reactionswere then increased to 68° C. for 2′. FIG. 19 shows that a nicetitration was observed for the different amounts of template DNA whilethe no template control (0 μM DNA) remained clean at 20 PCR cycles.Titration was observed for low concentrations of DNA template(concentration as low as 500 femtomolar DNA). This experiment alsosuggests that there may be inhibitory effects at high DNA templateconcentrations.

Sequences:

P188 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT CTTCCGATCTP173 (RNA template) CAGUCAGUCAGUCAGUCAGUGCCAAAUGCCUCGUCAUC P174TGATGACGAGGCATTTGGC

Example 12: Sensitivity Driver for Liquid Biopsy Application

This experiment shows DNA titration and sensitivity of up to 0.3 pg ofDNA. Similar protocol as previously described in the above examples(e.g., example 11) was followed with DNA template concentration of 0 μg,0.3 μg, 30 ng, 3 ng, 0.3 ng, 30 pg, 3 pg, or 0.3 pg (see, FIG. 20).Currently, none of the next generation sequencing sample preparationtechnology holds this capability for sensitivity. This experiment shows100-1000 fold higher sensitivity than any current available method. Datashows potential for applications with DNA with a few orders of magnitudelower than 0.3 pg.

Example 13: Template Concatemerization

This experiment was designed to demonstrate a method for convertingshort DNA fragments into a concatemer. Concatemers may contain about 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000,or more copies of the starting nucleic acid. A schematic of the workflowis shown in FIG. 24A. In brief, the initial PCR protocol for templatepreparation included H₂O, 2×Q5 master mix, P316 (0.5 μM), P317 (0.5 μM),and pUC18 (0.05 ng/4). The PCR condition was 98° C. for 30 secondsfollowed by 30 cycles of: 98° C. for 10 seconds, 66° C. for 15 seconds,and 72° C. for 10 seconds. At the end of the 30 cycles, the reaction waskept at 72° C. for 2 minutes and then reduced to 4° C. The adaptorannealing reaction included H₂O, Tris pH 8.0 (20 mM), NaCl (100 mM), andtwo primers (25 μM each; (P312+P313) or (P314+P315) or (P320+P321)). Thereaction was incubated at 90° C. for 1 minute, followed by 0.1°C./second ramp to 25° C. (20 seconds) and then reduced and kept at 4° C.The first adaptor ligation reaction included H₂O (30 μL), fragmented DNA(20 μL), end repair and T-tailing buffer (7 μL), and end repair andT-tailing enzyme mix (3 μL). The reaction was incubated at 20° C. for 30minutes and then increased to 65° C. for 30 minutes. H₂O (5 μL) was thenadded to the reaction (50 μL) along with 2.5 μL of 20 μM adaptor(P312+P313), 2.5 μL of 20 μM adaptor (P314+P315), ligation buffer (30μL), and DNA ligase (10 μL). The reaction was then incubated at roomtemperature for 15 minutes, followed by a reaction clean-up. SPRI beadswere added and the reaction was eluted. The adaptor ligated library (10μL) was incubated with H₂O (40 μL) and 2× Kappa HiFi master mix (50 μL)and subjected to PCR (98° C. for 45 seconds; 5 cycles of 98° C. for 15seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds; 72° C. for 1minute; and kept at 4° C.). This protocol can be modified in order toincrease the number of cycles (e.g., from 5 cycles to 25 cycles). Thesecond adaptor ligation reaction comprises of a similar protocol as theone described for the first adaptor ligation reaction; the differencebeing that 5 μL of 20 μM adaptor (P320+P321) was used instead of 2.5 μLof 20 μM adaptor (P312+P313) and 2.5 μL of 20 μM adaptor (P314+P315).The gel in FIG. 24B shows that the adapters were successfully ligated tothe fragmented DNA sequence and shows template concatemerization.

Sequences:

P312 ACACTCTTTCCCTACACGACGCT Right adaptor P313/5Phos/GCGTCGTGTAGGGAAAGAGTGT Right adaptor P314/5Phos/CACTCTTTCCCTACACGACGCT Left adaptor P315 AGCGTCGTGTAGGGAAAGAGTGTLeft adaptor P316 ACACTTTATGCTTCCGGCTC Amp pUC18 for 200 bp frag withKpnI in middle P317 TAAGTTGGGTAACGCCAGG Amp pUC18 for 200 bp frag withKpnI in middle P318 ACACTCTTTCC Invasion primers P319 AGCGTCGTGInvasion primers P320 TTCCAATGATACGGCGACCACCGAUACUGUCAUAGOutside adaptor can use P5 CTAGCTCCT primer USER compatible P321/5Phos/GGAGCTAGCTATGACAGTATCGGTGGTCGCC Outside adaptor-can use P5GTATCATTACTT primer

Example 14: Improved Conversion Efficiency (RNA Sample toNext-Generation Sequence (NGS) Library) after 3′-Phosphate, 2′-Phosphateand 2′, 3′-Cyclic Phosphate Removal

Some of the proposed or demonstrated techniques of the presentdisclosure require free 3′-hydroxyl at the 3′-end of an RNA sample. Forexample, 3′-OH is required for RNA poly(A) tailing with a polymerase(e.g., poly-A polymerase), and/or for DNA poly-tailing with terminaldeoxynucleotidyl transferase (TdT), and/or for ligation. Endogenous RNAusually contains a 3′-hydroxyl or a 2′,3′-cyclic phosphate or a3′-phosphate. The 3′-hydroxyl can be a product of transcription, poly(A)tail synthesis, or enzymatic cleavage (enzymes with catalytic mechanismsimilar to RNase H). The 2′,3′-cyclic phosphate can be a product ofenzymatic cleavage (enzymes like RNase A) or spontaneous hydrolysis(non-enzymatic intramolecular transphosphorylation). For example, RNAcan be cleaved by intramolecular transesterification.

The 2′,3′-cyclic phosphate is very common due to natural RNAphosphodiester bond instability and can occur naturally (cell free RNAdegradation) or as a result of sample treatment or storage. RNA samplesbearing 2′,3′-cyclic phosphate or 3′-phosphate cannot be subsequentlypoly-tailed or ligated because the presence of a free 3′-hydroxyl groupis required for both. For this reason, RNA samples with 2′,3′-cyclicphosphate or 3′-phosphate can be treated with a phosphatase (e.g., T4polynucleotide kinase (PNK) enzyme) to generate a 3′-hydroxyl group.Other examples of phosphatases are disclosed in TABLE 13 below (UshatiDas and Stewart Shuman, Mechanism of RNA 2′,3′-cyclic phosphate endhealing by T4 polynucleotide kinase-phosphatase, Nucleic Acids Research,2013, vol. 41, No. 1, 355-365).

TABLE 13 Comparison of RNA repair enzymes that heal 2′,3′-cyclicphosphate ends Enzyme Family Metal End-product CPDase product 3′-Pase2′-Pase T4 Pnkp Acylphosphatase Mg²⁺ 3′-OH, 2′-OH 3′-PO₄, 2′-OH Yes YesCthPnkp Binuclear metallophosphoesterase Mn²⁺ Ni²⁺ 3′-OH, 2′-OH 3′-OH,2′-PO₄ Yes Yes Yeast and plant tRNA ligase 2H phosphoesterase None3′-OH, 2′-PO₄ 3′-OH, 2′-PO₄ No No RtcB RtcB Mn²⁺ 3′-PO₄, 2′-OH 3′-PO₄,2′-OH No ?

T4 polynucleotide kinase (PNK) enzyme includes both kinase andphosphatase enzymatic activities. Thus, to optimize the T4 PNK, thekinase enzymatic activity can be removed by substituting at least one ofthe catalytically essential amino acids. This results in the phosphatasebeing the only enzymatic activity present. Removing the kinase activityhelps with subsequent reactions such as poly-A tailing using ATP forexample, because ATP is also a kinase substrate. Examples of cell freeRNA NGS library preparation protocols including de-phosphorylation aredisclosed herein. Also disclosed herein are comparison reactions (e.g.,reactions not treated with T4 PNK). The results showed a significant 3to 5-fold increase in the number of RNA particles captured in the NGSlibrary (refer to bio-analyzer trace data in FIG. 26).

Additional potential benefits: the unique properties of the 3′end of RNAparticles depending on the type of process used to generate the RNAparticles, allow one to focus and/or manipulate the sequencing library.For example, if one does not wish to sequence RNA fragments generateddue to process degradation (e.g., incomplete RNA fragments bearing2′,3′-cyclic phosphate), one can avoid treating the sample with T4 PNK.In this way, the library will include full mRNAs and miRNAs(3′-hydroxyl).

Example 15: RNA Sample Fragmentation is Part of the NGS LibraryPreparation Workflow; Enzymatic and Nonenzymatic Methods (Relevant forShort-Read Technologies Like Illumina and Ion Torrent)

Major DNA sequencing technologies, such as illumina or ion torrent, arelimited in regards to sequencing read-length (meaning that a limitednumber of bases can be sequenced in each individual read). Bothtechnologies have a read range of up to about 100 bp-500 bp, making itimpractical to use a library that significantly exceeds this range. Cellfree RNA usually ranges from about 20 to 2000 bases, formalin-fixedparaffin-embedded (FFPE) RNA ranges from about 20 to 500 bases and mRNAis usually around 2000 bases. For practical reasons, samples are usuallyfragmented, so effective library size is no more than 400 bp. Sampleloading library fragments longer than 1000 bp is very inefficientcompared to shorter fragments. Disclosed herein are two general methodsof RNA sample fragmentation; enzymatic and non-enzymatic. The enzymaticmethod can use enzymes with RNase activity (e.g., RNase A, RNase P,RNase H, RNase III, RNase T1, RNase T2, RNase U2, RNase V1, RNase I,RNase L, RNase PhyM, RNase V, dicer, or argonaute). The non-enzymaticmethod disclosed herein takes advantage of the natural chemicalinstability of RNAs. RNA can undergo spontaneous non-enzymaticfragmentation as a result of internal transphosphorylation. Breaking ofphosphodiester bonds of RNA can be brought about by various conditions(e.g., metals, such as Mg, Mn, Pb, or polyamines, or cofactors, such asPVP or PEG). An increase in the transphosphorylation rate can beachieved, for example, with high pH or with high(er) temperature.Non-enzymatic hydrolysis preferentially happens in single strandedportions of RNA particles, preferentially between bases UA or CA. Theadvantages of using a non-enzymatic method includes: simplicity andreliability (independent of enzyme activity or shelf life), and the factthat the reaction can be conducted in conditions compatible with themajority of the subsequent steps. TABLE 16 below shows a workflow ofboth a fragmentation protocol and a no-fragmentation protocol. FIG. 26shows the bioanalyzer trace data for the fragmentation protocol(represented by line 1) and the no-fragmentation protocol (representedby line 2). The libraries were prepared using cell free RNA sample.

TABLE 16 Workflow No Fragmentation With Fragmentation Work STEP_1 RNAfragmentation by flow: heat treatment STEP_1 PNK Treatment STEP_2 PNKTreatment STEP_2 Poly Adenylation STEP_3 Poly Adenylation using Poly-Ausing Poly-A Polymerase Polymerase STEP_3 Poly T Primer STEP_4 Poly TPrimer annealing annealing STEP_4 2D-RT & Tagging STEP_5 2D-RT & Taggingreaction reaction STEP_5 primer-adapter excess STEP_6 primer-adapterexcess and non-specific and non-specific priming product priming productcleaning cleaning with Magnetic beads with Magnetic beads withimmobilized with immobilized oligoA oligoA STEP_6 SPRI cleanup STEP_7SPRI cleanup STEP_7 Sample Index PCR STEP_8 Sample Index PCR STEP_8 SPRIcleanup STEP_9 SPRI cleanup

In short, the no-fragmentation protocol included 6.5 μL of H₂O, 2 μL of10×T4 PNK buffer, 0.5 μL of 10×RNase inhibitor, 1 μL of 10 U/4 T4 PNKenzyme, and 10 μL sample (e.g., cell free RNA sample). The reaction wasincubated at 37° C. for 20 minutes, 70° C. for 4 minutes, and thenplaced on ice. 3.25 μL of H₂O, 10 μL of 5×2D PNK buffer, 1.25 μL of10×RNase inhibitor, 7.5 μL of 10 mM ATP, and 1.25 μL of 5 U/4 E coliPolyA Pol were then added to the reaction. The reaction was incubated at16° C. for 5 minutes and then placed on ice. 0.5 μL of 100×dNTPs, 1 μLof 10 μM P334 Primer, 0.25 μL of 100 μM P423 DNA ter acc were then addedto the reaction. The reaction was incubated at 70° C. for 2 minutes, andthen placed on ice for 2 minutes. 1.25 μL of 10×RNase inhibitor, 3.75 μLof P2 (e.g., R2 variant at 1 μg/μL, (e.g., an R2 RT N-truncation, suchas SEQ ID NO: 50)) were added to the reaction (for a total of 50 μLreaction). The reaction was incubated at 34° C. for 1 hour, pulled down,spri 1.6×, then eluted in 50 μL. See FIG. 26, line 2. In some instances,a reverse transcriptase or a modified reverse transcriptase, or anenzyme that has similar function to a reverse transcriptase can be usedinstead of P2.

In short, the fragmentation protocol included 1 μL of 10× buffer A and 9μL of sample (e.g., cell free RNA sample). The reaction was incubated at94° C. for 4 minutes and then placed on ice. 14.75 μL of H₂O, 3 μL of10× buffer B, 0.75 μL of 10×RNase inhibitor, and 1.5 μL of 10 U/μL T4PNK enzyme were added to the reaction. The reaction was incubated at 37°C. for 30 minutes, at 72° C. for 3 minutes and then placed on ice. 5 μLof 10× buffer C, 1.25 μL of 10×RNase inhibitor, 7.5 μL of 10 mM ATP, and1.25 μL of 5 U/4 E coli PolyA Pol were then added to the reaction. Thereaction was incubated at 16° C. for 5 minutes and then placed on ice.0.5 μL of 100×dNTPs, 1 μL of 10 μM P334 Primer, 0.25 μL of 100 μM P423DNA ter acc were then added to the reaction. The reaction was incubatedat 70° C. for 2 minutes, and then placed on ice for 2 minutes. 1.25 μLof 10×RNase inhibitor and 3.75 μL of P2 (e.g., R2 variant at 1 μg/μL(e.g., an R2 RT N-truncation, such as SEQ ID NO: 50)) (were added to thereaction (for a total of 50 reaction). The reaction was incubated at 34°C. for 1 hour, pulled down, spri 1.6×, then eluted in 50 μL. See FIG.26, line 1. In some instances, a reverse transcriptase or a modifiedreverse transcriptase, or an enzyme that has similar function to areverse transcriptase can be used instead of P2.

In short, the 5×2D PNK buffer included 645 μL of H₂O, 10 μL of 1000 mMTris-HCl pH 7.5, 300 μL of 5000 mM NaCl₂, 5 μL of 1000 mM MgCl₂, 25 μLof 10% tween, and 15 μL of 1000 mM DTT. The buffer A stock included 60μL of H₂O, 10 μL of 1000 mM Tris-HCl pH 8.3, and 30 μL of 1000 mM MgCl₂.The buffer B stock included 45 μL of H₂O, 50 μL of 1000 mM Tris-HCl pH7.5, and 5 μL of 1000 mM DTT. The buffer C stock included 36 μL of H₂O,60 μL of 5000 mM NaCl₂, 2.5 μL of 10% tween, and 1.5 μL of 1000 mM DTT.The 10×PNK buffer included 150 μL of H₂O, 700 μL of 1000 mM Tris-HCl pH7.5, 100 μL of MgCl₂, and 50 μL of 1000 mM DTT. The 100× balanced dNTPsincluded 100 μL of H₂O, 75 μL of 100 mM dATP, 75 μL of 100 mM of dTTP,375 μL of 100 mM dGTP, and 375 μL of 100 mM dCTP. The 5×R2 buffer+dNTPsincluded 430 μL of H₂O, 150 μL of 1000 mM Tris-HCl pH 7.5, 300 μL of5000 mM NaCl₂, 25 μL of 1000 mM MgCl₂, 25 μL of 10% tween, 25 μL of 1000mM DTT, 3.75 μL of 100 mM dATP, 3.75 μL of 100 mM of dTTP, 18.75 μL of100 mM dGTP, and 18.75 μL of 100 mM dCTP. The streptavidin magneticbeads included 160 μL of streptavidin magnetic beads (NEB) saturatedwith biotinylated oligo AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA/3BioTEG/;beads were resuspended in 10 mM Tris pH7.5, 300 mM NaCl₂. The primersequences used were: P334 (A/iSp9/CCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN TTTTTTTTTTTTTTTTT); P423(AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCT/3ddC/); P399(AATGATACGGCGACCACCGAGATCTACACGTACTGACACACTCTTTCCCTACACGACGC); P400(CAAGCAGAAGACGGCATACGAGATATTACTCGGTGACTGGAGTTCAGACGTGT)

Example 16: Robust Mechanism of R2 RT Jumping

R2 RT jumping is a very efficient mechanism. It is much less sensitiveto the acceptor-adapter sequences compared to template switchingmechanisms (e.g., methods that use MMLV). This low sensitivity allowsfor optimal utilization of sequencing adapters in the Illuminasequencing for example. In this experiment, a variety of acceptors weretested. The results showed similar efficiency between RNA and DNAacceptors. The use of DNA acceptors allow for cheaper and more reliableand/or stable technology. The results also showed that the conversionefficiency was not sensitive to the 3′-end of the acceptor sequences.Thus, this mechanism allows for flexibility regarding acceptor sequencesand it is relevant for both RNA and DNA samples. Examples of acceptorsused: 1) AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGG/3ddC/; 2)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAGGG/3ddC/; 3)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTGGG/3ddC/; 4)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTG/3ddC/; 5)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCT/3ddC/; 6)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTrGrGrG/3ddC/; 7)AAAA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTrGrGrG; 8)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTN/3ddC/; 9)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN/3ddC/; 10)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCT*/3ddC/; 11)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATCTN/ideoxyl//3ddC/; 12)AA/iSp9/ACACTCTTTCCCTACACGACGCTCTTCCGATC/iSuper-dT//3ddC/; and 13)A/iSp9/CCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT/3ddC/.

Example 17: Poly-A Tail Length Control, Method with Non-ExtendableNucleotide

PolyA polymerase is an RNA polymerase used frequently to generate apoly-A tail on the 3′ end of an RNA (e.g., poly-A polymerase form E.coli or yeast). Poly-A polymerase has enzymatic activity that allows forthe generation of an RNA chain (i.e., extension of the 3′end of an RNA)without an RNA or DNA template. Although, poly-A polymerase preferablysynthesizes a poly-A tail, poly-A polymerase also has activity withother ribonucleotides (e.g., CTP, GTP and UTP). Controlling the poly-Atail length is important for sequencing quality and yield. Typically,ATP concentration and reaction time and/or temperature are used tocontrol the poly-A tail length. Alternative methods can be used, such asusing a blocking (un-extendable) nucleotide (e.g.,3′-Deoxyadenosine-5′-Triphosphate (an ATP analog)). Once a blockingnucleotide, 3′-Deoxyadenosine-5′-Triphosphate, is incorporated to an RNAchain analog, it cannot be further extended due to a lack of a 3′hydroxyl group. Various concentrations of ATP and3′-Deoxyadenosine-5′-Triphosphate were used. It was found that thepoly-A tail length could be controlled based on the concentration/ratioof ATP and 3′-Deoxyadenosine-5′-Triphosphate, which was independent ofreaction time and/or enzyme concentration. This method provides forsignificant protocol advantage when applied to high throughput orautomated processes.

Example 18: Methods of Generating cfDNA Library

Described herein are two methods of generating cfDNA libraries. To helpexplain both methods, the protocol was divided into two general steps.In the first step, an R2 enzyme (2D Genomics R2 enzyme and derivatives)and accessory enzymes were used. The product of this step includesdouble stranded DNA (dsDNA), such as one original sample strand flankedby attached adapters with known sequences. This dsDNA particle includesone original sample strand and one which is copied by R2 and accessoryenzymes. The copy strand includes degradable nucleotides, such as dUTP,which can be degraded upon treatment with Uracil-DNA Glycosylase (UDG)enzyme, for example. An alternative method to remove the copy strandincludes having dUTPs in the primer-adapter sequence or using anonsymmetrical adapter. In the second step, the original DNA strand withattached adapters is PCR amplified using high fidelity methods/enzymes(for example enzymes with proofread activity). At least one advantage ofretaining the original strand for sequencing is that it avoidsintroducing errors while copying it using a non-error correcting enzymesuch as an R2 enzyme. Hence, the resulting library is not compromisedfor fidelity.

Method 1 (see, FIG. 27): First, cell free DNA (cfDNA) is denatured toproduce a single-stranded DNA (ssDNA) sample. This allows for eachstrand to be analyzed separately compared to ligation based methods(both strands in one final product). Analyzing each strand separatelycreates more opportunities to capture rare mutations. The protocol canalso include a dephosphorylation step (3′ and 5′ ends). Next, ssDNA ismixed with a priming jumping complex including DNA or RNA adapter withannealed primer and R2 polymerase. The reaction includes dNTPs andcatalytic metals. In short, R2 first extends the primer on the adaptertemplate and then template jumps to the ssDNA sample. The generatedproduct is dsDNA with one nick between the adapter template and thessDNA sample. The next step is end repair with a DNA polymerase that has3′-to-5′ exonuclease activity (e.g., T4 pol, Klenov fragment, Bst DNApolymerase or Phi29 DNA pol). The polymerase may also posses DNA stranddisplacement capability. The resulting product is a dsDNA with bluntends or 3′ A-overhang. The nick may be replaced with a whole new strandwhere extension and displacement is primed by the 3′ hydroxyl of theoriginal sample strand. On the other hand, the nick can be repairedduring the next step ligation. Next step ligation includes an asymmetricadapter. Adapter is a DNA duplex with one single-stranded overhang atthe 5′end. This overhang includes a sequence complementary to at leastone of the PCR amplification primers, causing only one strand to beamplified. Alternatively, in order to isolate only the original ssDNAstrand, an adapter with both 3′ and 5′ overhangs can be used(Y-structure) in combination with dUTP and UDG degradation. PCRamplification is then performed and includes DNA primers that arespecific to both adapters. Only one of the strands from the previoussteps is amplified; the one including the original sample DNA. In someembodiments, isolation of the original ssDNA strand occurs between theend repair and the asymmetric adapter ligation steps.

Method 2 (see, FIG. 28): First, cell free DNA (cfDNA) is denatured toproduce single-stranded DNA (ssDNA) sample. The protocol can alsoinclude a dephosphorylation step (3′ and 5′ ends). Next, a polyA orpolyC tail is generated by, for example, the addition of terminaldeoxynucleotidyl transferase (TdT). The length of the TdT generated tailcan be controlled based on the combination of dATP and incubation timeor temperature (if polyA tailing was selected). Alternatively, thelength of the polyA tail can be controlled based on non-extendablenucleotides (e.g., ddATP or 3′-Deoxyadenosine-5′-Triphosphate). Here,the reaction includes both dATP and a non-extendable analog that wereused at various ratio and concentrations. Next, TdT can be temperaturekilled and the sample can be annealed with a primer-adaptercomplementary to the DNA tail sequence. The reaction is then mixed withdNTPs (including dUTP), catalytic metal, R2 enzyme, andacceptor-adapter. The R2 enzyme extends the annealed primer and jumps tothe acceptor-adapter to continue extending. The acceptor-adapterincludes a nucleotide block to prevent R2 from finishing the templateand jumping to another template. The product is a double-stranded DNAwith 5′ overhangs on both sides and a nick between a cfDNA sample strandand the acceptor-adapter. The product is then subject to end repair andnick ligation with a DNA polymerase having 3′-to-5′ exonucleolyticactivity and a DNA ligase (e.g., T4 pol, Klenov fragment, Bst DNApolymerase or Phi29 DNA pol, T4 DNA Ligase). The product of the endrepair and nick ligation is then subject to UDG degradation. The laststep is PCR amplification which includes DNA primers specific to bothadapters. Only one of the strands from the previous steps is amplified;the one including the original sample DNA.

Example 19: Library Preparation, Depletion of Ribosomal RNA (rRNA) andTransfer RNA (tRNA) to Maximize Sequencing Throughput

Approximately 80% of the total RNA in cells is rRNA and 15% is tRNA.Ribosomal RNA rarely serves as a diagnostic target. Therefore, becauseof that, the practice is to remove/deplete rRNA and tRNA from sequencinglibraries. The amount of rRNA and tRNA in sequencing libraries can becontrolled at various stages of library preparation. For example,depletion of rRNA and tRNA can occur during the early stages, e.g.,after total RNA isolation (RNA level), or after PCR amplification (dsDNAlevel). Two general methods to remove rRNA and tRNA is describedherein: 1) pulling rRNA/tRNA or PCR products using complementaryoligonucleotide attached to magnetic beads or solid support; and 2)oligonucleotide-guided degradation of the rRNA/tRNA or PCR products.

Method 1: In this method, amplified dsDNA is denatured and hybridized toa pool of strategically designed oligonucleotides. Oligonucleotides arecomplementary to one or both DNA strands with rDNA sequence. ForIllumina library, only one strand may be depleted as only one polarityis used in bridge amplification (see, FIG. 29). Each oligonucleotideincludes biotin modification. Ribosomal sequences (including DNAfragments) are depleted/removed using streptavidin-immobilized magneticbeads or solid support. In some cases, depletion is performed after PCRlibrary amplification in order to mitigate losses of rare and lowrepresented sequences. Depletion can also be performed during the earlystages of library preparation (e.g., RNA level).

Method 2: In this method, Cas9 nuclease is complexed with strategicallydesigned guiding RNA oligonucleotides (Carolin Anders and Martin Jinek,In vitro Enzymology of Cas9, Methods Enzymol., 2014; 546: 1-20).Oligonucleotides can have sequences complementary to ribosomal RNA.Complexes of Cas9 with guiding RNA oligonucleotide specifically bindsdsDNA (after PCR amplification) and rRNA sequences, and catalyzesendonucleolitic cleavage of single or both DNA strands (see, FIG. 30).

Embodiments

Embodiment 1. A method for preparing a complementary deoxyribonucleicacid (cDNA) molecule using template jumping, comprising: annealing aprimer to a template; and mixing, in the presence of nucleotides, thetemplate annealed to the primer with a modified reverse transcriptaseand an acceptor nucleic acid molecule under conditions sufficient togenerate a continuous cDNA molecule complementary to the template and tothe acceptor nucleic acid molecule, wherein the modified reversetranscriptase comprises at least one improved enzyme property relativeto a wild type or unmodified reverse transcriptase, wherein uponproducing the continuous cDNA molecule, the modified reversetranscriptase undergoes migration from the template to the acceptornucleic acid molecule.

Embodiment 2. A method for preparing a complementary deoxyribonucleicacid (cDNA) molecule using template jumping, comprising: (a) annealingone or more primer(s) to a template; and (b) mixing, in the presence ofnucleotides, the template annealed to one or more primer(s) with amodified reverse transcriptase and an acceptor nucleic acid moleculeunder conditions sufficient to generate a cDNA molecule complementary tothe template and/or to the acceptor nucleic acid molecule, wherein themodified reverse transcriptase comprises at least one improved enzymeproperty relative to a wild type or unmodified reverse transcriptase.

Embodiment 3. A method for preparing a nucleic acid molecule usingtemplate jumping comprising: mixing, in the presence of nucleotides, afragment or degraded template, a primer, a modified reversetranscriptase, and an acceptor nucleic acid molecule under conditionssufficient to generate a nucleic acid molecule, wherein the modifiedreverse transcriptase comprises at least one improved enzyme propertyrelative to a wild type or unmodified reverse transcriptase.

Embodiment 4. A method for preparing a nucleic acid molecule usingtemplate jumping comprising: mixing, in the presence of nucleotides, afragment or degraded template, a donor complex, a modified reversetranscriptase, and an acceptor nucleic acid molecule under conditionssufficient to generate a nucleic acid molecule, wherein the modifiedreverse transcriptase comprises at least one improved enzyme propertyrelative to a wild type or unmodified reverse transcriptase.

Embodiment 5. A method for preparing a complementary deoxyribonucleicacid (cDNA) library using template jumping, the method comprising: (a)annealing one or more primer(s) to a template; (b) mixing, in thepresence of nucleotides, the template annealed to one or more primer(s)with an acceptor nucleic acid molecule and a modified reversetranscriptase, under conditions sufficient to generate a continuous cDNAmolecule complementary to the template and/or to the acceptor nucleicacid molecule, wherein the modified reverse transcriptase comprises atleast one improved enzyme property relative to a wild type or unmodifiedreverse transcriptase, wherein upon producing the continuous cDNAmolecule, the modified reverse transcriptase undergoes migration fromthe template to the acceptor nucleic acid molecule; and (c) amplifyingthe cDNA molecule to generate a cDNA library.

Embodiment 6. A method for preparing a complementary deoxyribonucleicacid (cDNA) library using template jumping, the method comprising: (a)mixing, in the presence of nucleotides, a fragmented or degradedtemplate with a primer, a modified reverse transcriptase, and anacceptor nucleic acid molecule under conditions sufficient to generate acDNA molecule, wherein the modified reverse transcriptase comprises atleast one improved enzyme property relative to a wild type or unmodifiedreverse transcriptase; and (b) amplifying the cDNA molecule to generatea cDNA library.

Embodiment 7. A method for preparing a deoxyribonucleic acid (DNA)library using template jumping, the method comprising: (a) mixing afragmented or degraded template with a primer, a modified reversetranscriptase, and an acceptor nucleic acid molecule under conditionssufficient to generate a DNA molecule, wherein the modified reversetranscriptase comprises at least one improved enzyme property relativeto a wild type or unmodified reverse transcriptase; and (b) amplifyingthe DNA molecule to generate a DNA library.

Embodiment 8. A method for preparing a complementary deoxyribonucleicacid (cDNA) library using template jumping, the method comprising: (a)mixing a fragmented or degraded template with a donor complex, amodified reverse transcriptase, and an acceptor nucleic acid moleculeunder conditions sufficient to generate a cDNA molecule, wherein themodified reverse transcriptase comprises at least one improved enzymeproperty relative to a wild type or unmodified reverse transcriptase;and (b) amplifying the cDNA molecule to generate a cDNA library.

Embodiment 9. A method for preparing a deoxyribonucleic acid (DNA)library using template jumping, the method comprising: (a) mixing afragmented or degraded template with a donor complex, a modified reversetranscriptase, and an acceptor nucleic acid molecule under conditionssufficient to generate a DNA molecule, wherein the modified reversetranscriptase comprises at least one improved enzyme property relativeto a wild type or unmodified reverse transcriptase; and (b) amplifyingthe DNA molecule to generate a DNA library.

Embodiment 10. A method for preparing a library for sequencingcomprising: (a) obtaining a sample with cell-free nucleic acid from asubject; (b) mixing a modified reverse transcriptase enzyme, a template,nucleotides, an acceptor nucleic acid molecule, and one or moreprimer(s) to the cell-free nucleic acid, wherein the modified reversetranscriptase is capable of template jumping and comprises at least oneimproved enzyme property relative to a wild type or unmodified reversetranscriptase; (c) conducting an amplification reaction on cell-freenucleic acid (cf nucleic acid) derived from the sample to produce aplurality of amplicons, wherein the amplification reaction comprises 35or fewer amplification cycles; and (d) producing a library forsequencing, the library comprising a plurality of amplicons.

Embodiment 11. A method for preparing a complementary deoxyribonucleicacid (cDNA) library from a plurality of single cells, the methodcomprising the steps of: (a) releasing nucleic acid from each singlecell to provide a plurality of individual nucleic acid samples, whereinthe nucleic acid in each individual nucleic acid sample is from a singlecell; (b) annealing the nucleic acid template to one or more primer(s);(c) mixing, in the presence of nucleotides, the nucleic acid templateannealed to one or more primer(s) with an acceptor nucleic acid moleculeand a modified reverse transcriptase under conditions effective forproducing a cDNA molecule, wherein the modified reverse transcriptase iscapable of template jumping and comprises at least one improved enzymeproperty relative to a wild type or unmodified reverse transcriptase;and (d) amplifying the cDNA molecule to generate a cDNA library.

Embodiment 12. A method for preparing a deoxyribonucleic acid (DNA)library from a plurality of single cells, the method comprising thesteps of: (a) releasing nucleic acid from each single cell to provide aplurality of individual nucleic acid samples, wherein the nucleic acidin each individual nucleic acid sample is from a single cell; (b)annealing the nucleic acid template to one or more primer(s); (c)mixing, in the presence of nucleotides, the nucleic acid templateannealed to one or more primer(s) with an acceptor nucleic acid moleculeand a modified reverse transcriptase under conditions effective forproducing a DNA molecule, wherein the modified reverse transcriptase iscapable of template jumping and comprises at least one improved enzymeproperty relative to a wild type or unmodified reverse transcriptase;and (d) amplifying the DNA molecule to generate a DNA library.

Embodiment 13. A method for detecting a nucleic acid molecule, themethod comprising the steps of: (a) mixing a sample comprising a nucleicacid molecule with an acceptor nucleic acid molecule, a modified reversetranscriptase, a primer, and nucleotides, under conditions effective forgenerating a nucleic acid molecule, wherein the modified reversetranscriptase comprises at least one improved enzyme property relativeto a wild type or unmodified reverse transcriptase; and (b) amplifyingthe nucleic acid molecule.

Embodiment 14. A method for determining the presence of cancer in asubject comprising: (a) obtaining a biological sample from a subject;(b) detecting a nucleic acid molecule in the biological sample ofembodiment 13; (c) sequencing the nucleic acid molecule; and (d)determining that the subject is afflicted with cancer based on thepresence of the nucleic acid molecule.

Embodiment 15. A method for preparing a complementary deoxyribonucleicacid (cDNA) molecule using template jumping, comprising mixing, in asingle tube, a primer or one or more primer(s), a messenger RNA (mRNA)template, nucleotides, a modified reverse transcriptase, an acceptornucleic acid molecule, and a catalytic metal under conditions sufficientto generate a continuous cDNA molecule complementary to the mRNAtemplate and to the acceptor nucleic acid molecule, wherein the modifiedreverse transcriptase comprises at least one improved enzyme propertyrelative to a wild type or unmodified reverse transcriptase, whereinupon producing the continuous cDNA molecule, the modified reversetranscriptase undergoes migration from the template to the acceptornucleic acid molecule.

Embodiment 16. A method for preparing a library for sequencingcomprising mixing, in a single tube, a cell-free nucleic acid, amodified reverse transcriptase enzyme, a template, nucleotides, anacceptor nucleic acid molecule, a catalytic metal, and one or moreprimer(s), under conditions sufficient to generate a library, whereinthe modified reverse transcriptase comprises at least one improvedenzyme property relative to a wild type or unmodified reversetranscriptase.

Embodiment 17. A method for preparing a library under conditionssufficient to generate a library for sequencing comprising: (a)obtaining a sample with cell-free nucleic acid from a subject; (b)mixing a modified reverse transcriptase, a template, nucleotides, andone or more primer(s) to the cell-free nucleic acid; (c) conducting anamplification reaction on cell-free nucleic acid (cf nucleic acid)derived from a sample to produce a plurality of amplicons, wherein theamplification reaction comprises 35 or fewer amplification cycles; and(d) producing a library for sequencing, the library comprising theplurality of amplicons.

Embodiment 18. A method for preparing a cDNA molecule comprising mixing,in the presence of nucleotides, a primer, a template, a modified reversetranscriptase and an acceptor nucleic acid molecule under conditionssufficient to generate a cDNA molecule complementary to the templateand/or to the acceptor nucleic acid molecule, wherein the modifiedreverse transcriptase comprises at least one improved enzyme propertyrelative to a wild type or unmodified reverse transcriptase.

Embodiment 19. A method for preparing a cDNA molecule comprising mixing,in the presence of nucleotides, one or more primer(s), a template, amodified reverse transcriptase, and an acceptor nucleic acid moleculeunder conditions sufficient to generate a cDNA molecule complementary tothe template and/or to the acceptor nucleic acid molecule, wherein themodified reverse transcriptase comprises at least one improved enzymeproperty relative to a wild type or unmodified reverse transcriptase.

Embodiment 20. A method for preparing a cDNA library comprising mixing,in the presence of nucleotides, a primer or one or more primer(s), atemplate, a modified reverse transcriptase, and an acceptor nucleic acidmolecule under conditions sufficient to generate a cDNA moleculecomplementary to the template and/or to the acceptor nucleic acidmolecule, wherein the modified reverse transcriptase comprises at leastone improved enzyme property relative to a wild type or unmodifiedreverse transcriptase; and amplifying the cDNA molecule to generate acDNA library.

Embodiment 21. The method of any one of embodiments 1-20, wherein thenucleic acid molecule (e.g., the acceptor nucleic acid molecule or thecell free nucleic acid molecule) or the template comprises an unknownnucleic acid sequence.

Embodiment 22. The method of any one of embodiments 1, 5, and 15,wherein the migration is independent of sequence identity between thetemplate and the acceptor nucleic acid molecule.

Embodiment 23. The method of any one of embodiments 1-20, wherein themixing comprises addition of a hot start thermostable polymerase.

Embodiment 24. The method of embodiment 23, wherein the hot startthermostable polymerase is a hot start taq polymerase.

Embodiment 25. The method of any one of embodiments 1-20, wherein themethod is performed in a single tube (single vessel).

Embodiment 26. The method of any one of embodiments 1-20, furthercomprising performing a polymerase chain reaction (PCR) amplificationreaction.

Embodiment 27. The method of embodiment 26, wherein the PCRamplification reaction is performed in the same single tube (samevessel) as the single tube (single vessel) of embodiment 25.

Embodiment 28. The method of any one of embodiments 1-16, and 18-20,wherein the acceptor nucleic acid molecule comprises a modifiednucleotide that causes primer extension to stop.

Embodiment 29. The method of any one of embodiments 10, 16, and 17,wherein the cell-free nucleic acid is cell-free DNA (cfDNA), circulatingtumor DNA (ctDNA), or formalin-fixed, paraffin-embedded DNA (FFPE DNA).

Embodiment 30. The method of embodiment 26, wherein the PCRamplification is performed at a temperature sufficient to inactivate thereverse transcriptase.

Embodiment 31. The method of embodiment 26, wherein the PCRamplification is performed at a temperature sufficient to activate thehot start thermostable polymerase.

Embodiment 32. The method of any one of the embodiments 1-16, and 18-20,wherein the acceptor nucleic acid molecule is modified at the 3′ end.

Embodiment 33. The method of embodiment 32, wherein the modificationcomprises a dideoxy 3′ end and/or a phosphorylated 3′ end.

Embodiment 34. The method for preparing a nucleic acid molecule and/orlibrary or a complementary cDNA molecule and/or library of any one ofembodiments 1-20, wherein the molecule and/or library is prepared in atmost about 2 hours.

Embodiment 35. The method of any one of embodiments 10-14 and 17,wherein the sample comprises a circulating tumor DNA sample or a tissuesample.

Embodiment 36. The method of any one of embodiments 1-20, wherein thenucleic acid molecule and/or the acceptor nucleic acid molecule is RNA,DNA, or a combination of RNA and DNA.

Embodiment 37. The method of any one of embodiments 1-12 and 15-20,wherein the template is RNA, DNA, or a combination of RNA and DNA.

Embodiment 38. The method of any one of embodiments 4, 8, and 9, whereinthe donor complex comprises a template and a primer.

Embodiment 39. The method of embodiment 37, wherein the template is afragmented or degraded template.

Embodiment 40. The method of any one of embodiments 36 and 37, whereinthe RNA is mRNA.

Embodiment 41. The method of any one of embodiments 1-20, furthercomprising depleting ribosomal RNA (rRNA) and/or transfer RNA (tRNA).

Embodiment 42. The method of embodiment 41, wherein the step fordepleting rRNA comprises hybridization of an oligonucleotide to an rRNA.

Embodiment 43. The method of any one of embodiments 1-20, wherein thenucleic acid and/or the template is from a sample.

Embodiment 44. The method of any one of embodiments 10-14, 17, and 43,wherein the sample is a liquid biopsy sample.

Embodiment 45. The method of any one of embodiments 1-20, wherein thenucleic acid and/or the template is equal to or less than about 0.01micromolar.

Embodiment 46. The method of embodiment 45, wherein the nucleic acidand/or the template is equal to or less than about 500 femtomolar.

Embodiment 47. The method of any one of embodiments 1-20, wherein thenucleic acid and/or the template is a fragment or a degraded nucleicacid, a fragmented DNA, a fragmented RNA, or any combination thereof.

Embodiment 48. The method of embodiment 13, wherein the nucleic acidmolecule is indicative of a disease.

Embodiment 49. The method of embodiment 48, wherein the disease iscancer.

Embodiment 50. The method of any one of embodiments 1-20, furthercomprising providing a prenatal diagnosis based on the presence orabsence of a nucleic acid molecule.

Embodiment 51. The method of any one of embodiments 1-20, furthercomprising optimization of the template, wherein the optimizationcomprises contacting a sample comprising the template with an agent thatremoves the 5′ cap structure of the template, under conditionspermitting the removal of the cap structure by the agent, therebyforming a decapped template.

Embodiment 52. The method of embodiment 51, further comprisingcontacting the sample with a dephosphorylating agent under conditionspermitting the dephosphorylation of the decapped template by the agent.

Embodiment 53. The method of embodiment 51, wherein the agent ispyrophosphatase.

Embodiment 54. The method of embodiment 52, wherein the agent isalkaline phosphatase.

Embodiment 55. The method of any one of embodiments 1-20, wherein theprimer is fluorescently labeled.

Embodiment 56. The method of any one of embodiments 1-20, wherein thereverse transcriptase is a non-long terminal repeat (LTR)retrotransposon (e.g., wherein the modified reverse transcriptase is anon-long terminal repeat (LTR) retrotransposon).

Embodiment 57. The method of any one of embodiments 1-20, wherein thereverse transcriptase is an R2 reverse transcriptase (e.g., wherein themodified reverse transcriptase is a modified R2 reverse transcriptase).

Embodiment 58. The method of embodiment 56, wherein the non-LTRretrotransposon is an R2 non-LTR retrotransposon.

Embodiment 59. The method of any one of embodiments 1-12 and 15, whereinthe template jumping is dependent on the concentration of the acceptornucleic acid molecule.

Embodiment 60. The method of any one of embodiments 1-16 and 18-20,wherein the improved enzyme property comprises at least one of thefollowing: increased stability (e.g., increased thermostability);increased specific activity; increased protein expression; improvedpurification; improved processivity; improved strand displacement;improved end-to-end template jumping; increased DNA/RNA affinity; andincreased fidelity.

Embodiment 61. The method of any one of embodiments 1-20, wherein themodified reverse transcriptase comprises an N-terminal truncation, aC-terminal truncation, or N-terminal and C-terminal truncations.

Embodiment 62. The method of any one of embodiments 1-20, wherein themodified reverse transcriptase comprises a truncation of less than about500 amino acid residues.

Embodiment 63. The method of any one of embodiments 1-20, wherein themodified reverse transcriptase comprises one or more of the followingmodifications: (a) an amino-terminal truncation of less than about 400amino acid residues; and (b) a carboxyl-terminal truncation of less thanabout 400 amino acid residues.

Embodiment 64. The method of any one of the embodiments 1-20, whereinthe modified reverse transcriptase further comprises a tag.

Embodiment 65. The method of any one of embodiments 1, 2, 5, 6, 8, 11,15, and 18-20, wherein the cDNA molecule further comprises a tag.

Embodiment 66. The method of any one of embodiments 1-12 and 15-20,wherein the template further comprises a tag.

Embodiment 67. The method of any one of embodiments 64-66, furthercomprising sequencing the modified reverse transcriptase comprising atag, or the cDNA molecule comprising a tag, or the template comprising atag.

Embodiment 68. The method of embodiment 67, wherein the sequencingcomprises whole transcriptome analysis.

Embodiment 69. The method of any one of embodiments 64-66, wherein thetag is at least one member selected from the group consisting of biotin,azido group, acetylene group, His-tag, Calmodulin-tag, CBP, CYD, StrepII, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag-1, Softag-3,V5-tag, Xpress-tag, Isopeptag, SpyTag, B, HPC peptide tags, GST, MBP,biotin, biotin carboxyl carrier protein, glutathione-S-transferase-tag,green fluorescent protein-tag, maltose binding protein-tag, Nus-tag,Strep-tag, and thioredoxin-tag.

Embodiment 70. The method of any one of embodiments 1-20, furthercomprising purifying a solution comprising the continuous cDNA molecule,or the nucleic acid molecule, or the DNA molecule.

Embodiment 71. The method of any one of embodiments 1-20, wherein themethod is performed in the absence of purification.

Embodiment 72. The method of any one of embodiments 1-20, wherein themodified reverse transcriptase is derived from an arthropod.

Embodiment 73. The method of embodiment 72, wherein the arthropod isBombyx mori.

Embodiment 74. The method of any one of embodiments 1-20, wherein themodified reverse transcriptase is purified.

Embodiment 75. The method of embodiment 74, wherein the modified reversetranscriptase is at least about 80% pure.

Embodiment 76. The method of embodiment 70, wherein the purifying asolution comprises two purification steps.

Embodiment 77. The method of embodiment 76, wherein the two purificationsteps comprise a nickel and a heparin affinity purification.

Embodiment 78. The method of embodiment 74, wherein the modified reversetranscriptase is purified by immobilized metal affinity chromatography(IMAC).

Embodiment 79. The method of any one of embodiments 1-20, wherein themethod further comprises a salt.

Embodiment 80. The method of embodiment 79, wherein the salt is at leastone member selected from the group consisting of NaCl, LiCl, AlCl₃,CuCl₂, MgCl₂, InCl₃, SnCl₄, CrCl₂, CrCl₃, KCl, NaI, KI, TMACl(tetramethyl ammonium chloride), TEACl (tetraethyl ammonium chloride),KSCN, CsSCN, KCH₃COO, CH₃COONa, C₅H₈KNO₄, C₅H₈NNaO₄, CsCl, and anycombination thereof.

Embodiment 81. The method of embodiment 80, wherein the salt comprisesNaCl.

Embodiment 82. The method of any one of embodiments 1-20, wherein themethod further comprises a detergent.

Embodiment 83. The method of embodiment 82, wherein the detergent is anon-ionic and/or zwitterionic detergent.

Embodiment 84. The method of embodiment 83, wherein the non-ionicdetergent is selected from a group consisting of tween, triton, TritonCF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-SM, TritonN-101 (Polyoxyethylene branched nonylphenyl ether), Triton QS-15, TritonQS-44, Triton RW-75 (Polyethylene glycol 260 monoChexadecyl/octadecyl)ether and 1-Octadecanol), Triton X-100 (Polyethylene glycoltert-octylphenyl ether), Triton X-102, Triton X-15, Triton X-151, TritonX-200, Triton X-207, Triton X-114, Triton X-165, Triton X-305, TritonX-405 (polyoxyethylene(40) isooctylphenyl ether), Triton X-405 reduced(polyoxyethylene(40) isooctylcyclohexyl ether), Triton X-45(Polyethylene glycol 4-tert-octylphenyl ether), Triton X-705-70, TWEENin any form including: TWEEN 20 (Polyoxyethylene sorbitan monolaurate),TWEEN 21 (Polyoxyethylene sorbitan monolaurate), TWEEN 40(polyoxyethylene(20) sorbitan monopalmitate), TWEEN 60 (Polyethyleneglycol sorbitan monostearate), TWEEN 61 (Polyethylene glycol sorbitanmonostearate), TWEEN 65 (Polyoxyethylene sorbitan Tristearate), TWEEN 80(Polyoxyethylene sorbitan monooleate), TWEEN 81 (Polyoxyethylenesorbitan monooleate), TWEEN 85 (polyoxyethylene(20) sorbitan trioleate),Brij, Brij 30 (Polyoxyethylene 4 lauryl ether) Brij 35 (Polyoxyethylene23 lauryl ether), Brij 52 (Polyoxyethylene 2 cetyl ether), Brij56(Polyoxyethylene 10 cetyl ether), Brij 58 (Polyoxyethylene 20 cetylether), Brij 72 (Polyoxyethylene 2 stearyl ether), Brij 76(Polyoxyethylene 10 stearyl ether), Brij 78 (Polyoxyethylene 20 stearylether), Brij 92 (Polyoxyethylene 2 oleyl ether), Brij 97(Polyoxyethylene 10 oleyl ether), Brij 98 (Polyoxyethylene 20 oleylether), Brij700 (Polyoxyethylene 100 stearyl ether, octyl thioglucoside,maltosides, and combinations thereof.

Embodiment 85. The method of any one of embodiments 79-84, wherein thesalt or the detergent improves enzyme activity or template jumping.

Embodiment 86. The method of any one of embodiments 5-13, 17, and 20,wherein the amplification step comprises PCR.

Embodiment 87. The method of embodiment 86, wherein the PCR comprises atleast one amplification primer and/or a polymerase.

Embodiment 88. The method of embodiment 87, wherein the PCR comprisesabout 4-8 cycles, about 10-40 cycles, or about 1-15 cycles.

Embodiment 89. The method of embodiment 86, wherein the PCR comprisesabout 30 cycles.

Embodiment 90. The method of embodiment 87, wherein the polymerase is ahot start polymerase.

Embodiment 91. The method of embodiment 86, further comprising detectingan amplicon generated by the amplification primers, wherein the presenceof the amplicon determines whether the modified reverse transcriptase ispresent in the sample.

Embodiment 92. The method of any one of embodiments 1-20, furthercomprising a temperature of about 12° C. to about 42° C. for about 1minute to about 5 hours.

Embodiment 93. The method of any one of embodiments 1-20, wherein themethod is carried out as a one-pot (single vessel) reaction.

Embodiment 94. The method of any one of embodiments 1-12 and 15-20,wherein the template is between about 30 base pairs and about 15000 basepairs.

Embodiment 95. The method of embodiment 94, wherein the template isabout 200 base pairs, or about 600 base pairs.

Embodiment 96. The method of any one of embodiments 1-12 and 15-20,wherein the template is between about 0.0001 micromolar and about 0.1micromolar.

Embodiment 97. The method of embodiment 96, wherein the template is atleast about 0.0001 micromolar.

Embodiment 98. The method of any one of embodiments 1-12 and 15-20,wherein the template or fragmented or degraded template is at leastabout 50 femtomolar.

Embodiment 99. The method of any one of embodiments 1-13, 15-16, and18-20, wherein the acceptor nucleic acid molecule comprises at least onemodified nucleotide.

Embodiment 100. The method of any one of embodiments 1-20, wherein theprimer comprises one or more random primer(s).

Embodiment 101. The method of any one of embodiments 1-20, wherein theprimer comprises an R2 RNA primer.

Embodiment 102. The method of any one of embodiments 1-20, wherein theprimer further comprises an adapter sequence.

Embodiment 103. The method of any one of embodiments 1-20, wherein themodified reverse transcriptase, comprises a fusion with Fhb, MBP, NusA,Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr,Ecotin, CaBP, ArsC, IF2-domain I, an expressivity tag, RpoA, SlyD, Tsf,RpoS, PotD, Crr, msyB, yjgD, rpoD, or any combination thereof.

Embodiment 104. A method for preparing a modified reverse transcriptase,which method comprises at least one of the following steps: (a)subjecting a DNA sequence encoding a reverse transcriptase enzyme torandom or rational mutagenesis; (b) subjecting a DNA sequence encoding areverse transcriptase enzyme to truncation of amino acids; (c)subjecting a DNA sequence encoding a reverse transcriptase enzyme toalteration comprising an insertion, a deletion or a substitution of anamino acid residue; (d) subjecting a DNA sequence encoding a reversetranscriptase enzyme to fusion with a protein or domain; and (e)subjecting a DNA sequence encoding a reverse transcriptase enzyme tohomologous genes DNA shuffling; wherein the DNA sequence obtained in anyone of steps (a) to (e) is expressed in a host cell, and wherein themodified reverse transcriptase comprises at least one improved enzymeproperty relative to a wild type or unmodified reverse transcriptase.

Embodiment 105. The method of embodiment 104, further comprisingscreening for host cells expressing the modified reverse transcriptase.

Embodiment 106. The method of embodiment 105, further comprisingpreparing the modified reverse transcriptase expressed by the hostcells.

Embodiment 107. The method of embodiment 104, further comprising atleast one of the following: determining the reverse transcriptase (RT)activity, estimating the reverse transcriptase active fraction(s), andtesting the stability and robustness of the mutants.

Embodiment 108. The method of embodiment 107, wherein the RT activity,the RT active fraction(s), or the stability and robustness of themutants are monitored via a reverse transcriptase activity assay.

Embodiment 109. A modified polypeptide having reverse transcriptaseactivity and at least one altered characteristic that improves enzymeproperty, wherein the modified polypeptide comprises an amino acidsequence having at least about 85% amino acid sequence identity to anyone of the sequences corresponding to National Center for BiotechnologyInformation (NCBI) GenBank database Accession Numbers provided in TABLE1.

Embodiment 110. A modified polypeptide having reverse transcriptaseactivity and at least one altered characteristic relative to a wild typeor unmodified reverse transcriptase, which altered characteristicenables the reverse transcriptase to (i) generate a complementarydeoxyribonucleic acid (cDNA) molecule from a template nucleic acidmolecule without thermal cycling, and (ii) generate one or more copiesof the cDNA molecule at an error rate of at most about 5%, wherein themodified polypeptide is a truncated variant of any one of the sequencescorresponding to any one of the Accession Numbers provided in TABLE 1.

Embodiment 111. A modified polypeptide having reverse transcriptaseactivity and at least one altered characteristic that improves enzymeproperty, wherein the modified polypeptide comprises an amino acidsequence having at least about 85% amino acid sequence identity to SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67.

Embodiment 112. The modified polypeptide of any one of embodiments109-111, wherein the characteristic that improves enzyme propertycomprises at least one of the following: increased stability; increasedspecific activity; increased protein expression; improved purification;improved processivity; improved strand displacement; improved templatejumping; increased DNA/RNA affinity; and increased fidelity.

Embodiment 113. The modified polypeptide of any one of embodiments109-111, wherein the modified polypeptide comprises an N-terminaltruncation, a C-terminal truncation, or N-terminal and C-terminaltruncations.

Embodiment 114. The modified polypeptide of any one of embodiments109-113, wherein the modified polypeptide comprises a truncation of lessthan about 100 amino acid residues.

Embodiment 115. The modified polypeptide of any one of embodiments109-114, wherein the modified polypeptide comprises one or more of thefollowing modifications: (a) an amino-terminal truncation of less thanabout 400 amino acid residues; and (b) a carboxyl-terminal truncation ofless than about 400 amino acid residues.

Embodiment 116. The modified polypeptide of any one of embodiments109-115, wherein the modified polypeptide comprises a fusion partner ora carrier protein.

Embodiment 117. The modified polypeptide of embodiment 116, wherein themodified polypeptide comprises a fusion with Fhb, MBP, NusA, Trx, SUMO,GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr, Ecotin, CaBP,ArsC, IF2-domain I, an expressivity tag, RpoA, SlyD, Tsf, RpoS, PotD,Crr, msyB, yjgD, rpoD, or any combination thereof.

Embodiment 118. The modified polypeptide of embodiment 116, wherein thefused variant comprises at least one of the following: increased shelflife, increased active fraction(s), and improved purification ascompared to the non-fused polypeptide.

Embodiment 119. A non-naturally occurring enzyme that subjects atemplate nucleic acid molecule to reverse transcription to generate acomplementary deoxyribonucleic acid (cDNA) product and amplification ofthe cDNA product at a processivity of at least about 80% per base asmeasured at 30° C.

Embodiment 120. A non-naturally occurring enzyme that subjects atemplate nucleic acid molecule to reverse transcription to generate anucleic acid product and amplification of the nucleic acid product at aprocessivity of at least about 80% per base as measured at 30° C.

Embodiment 121. The non-naturally occurring enzyme of embodiment119-120, wherein the non-naturally occurring enzyme has a performanceindex greater than 1.0 for at least one enzyme property.

Embodiment 122. The non-naturally occurring enzyme of embodiment 121,wherein the enzyme property is selected from the group consisting ofimproved stability, specific activity, protein expression, purification,processivity, strand displacement, template jumping, increased DNA/RNAaffinity, and fidelity.

Embodiment 123. A non-naturally occurring enzyme that subjects atemplate nucleic acid molecule to reverse transcription to generate acomplementary deoxyribonucleic acid (cDNA) product and amplification ofthe cDNA product in a time period of 3 hours or less at a performanceindex greater than 1.0 for at least one enzyme property selected fromthe group consisting of improved stability, specific activity, proteinexpression, purification, processivity, strand displacement, templatejumping, increased DNA/RNA affinity, and fidelity, as measured at atemperature from about 12° C. to about 42° C.

Embodiment 124. A non-naturally occurring enzyme that subjects atemplate nucleic acid molecule to reverse transcription to generate anucleic acid product and amplification of the nucleic acid product in atime period of 3 hours or less at a performance index greater than 1.0for at least one enzyme property selected from the group consisting ofimproved stability, specific activity, protein expression, purification,processivity, strand displacement, template jumping, increased DNA/RNAaffinity, and fidelity, as measured at a temperature from about 12° C.to about 42° C.

Embodiment 125. The non-naturally occurring enzyme of any one ofembodiments 123-124, wherein the template nucleic acid molecule is acell-free nucleic acid molecule.

Embodiment 126. A non-naturally occurring enzyme that subjects atemplate nucleic acid molecule to reverse transcription to generate acomplementary deoxyribonucleic acid (cDNA) product and amplification ofthe cDNA product in a time period of about 3 hours or less at aprocessivity for a given nucleotide substrate that is at least about 5%higher than the processivity of a reference enzyme for the samenucleotide substrate.

Embodiment 127. A non-naturally occurring enzyme that subjects atemplate nucleic acid molecule to reverse transcription to generate anucleic acid product and amplification of the nucleic acid product in atime period of about 3 hours or less at a processivity for a givennucleotide substrate that is at least about 5% higher than theprocessivity of a reference enzyme for the same nucleotide substrate.

Embodiment 128. A method of amplifying a nucleic acid molecule,comprising subjecting the nucleic acid molecule to nucleic acidamplification using a modified reverse transcriptase, wherein thereverse transcriptase is capable of amplifying the nucleic acid moleculeat processivity of at least about 80% per base at about 30° C.

Embodiment 129. The method of embodiment 128, further comprising usingthe modified reverse transcriptase to subject a template nucleic acidmolecule to reverse transcription to yield the nucleic acid molecule.

Embodiment 130. The method of embodiment 128, wherein the nucleic acidmolecule is a cell-free nucleic acid molecule.

Embodiment 131. A kit comprising: a primer, one or more primer(s), or arandom primer, nucleotides, at least one modified reverse transcriptase,a template, and instructions to perform the method of any one ofembodiments 1-20.

Embodiment 132. The kit of embodiment 131, wherein the kit comprises amodified reverse transcriptase that has reverse transcription or nucleicacid amplification activity and is capable of template jumping at atemperature of about 30° C.

Embodiment 133. A kit for detecting nucleic acid comprising a template,at least one modified reverse transcriptase, nucleotides, andinstructions to perform the method of any one of embodiments 3-4 and 13,wherein the nucleic acid is present at a concentration of at least about50 femtomolar.

Embodiment 134. A method of detecting, diagnosing, or prognosing acancer in a subject in need thereof, the method comprising: (a)obtaining sequence information of a cell-free nucleic acid samplederived from the subject of embodiment 10; and (b) using the sequenceinformation derived from (a) to detect a tumor nucleic acid in thecell-free nucleic acid sample, wherein the tumor nucleic acid isdetected at a concentration that is less than or equal to about 2% oftotal nucleic acid molecules in the cell-free nucleic acid sample.

Embodiment 135. The method of embodiment 134, wherein the tumor nucleicacid is detected at a concentration that is less than or equal to about1.75% of total nucleic acid molecules in the cell-free nucleic acidsample.

Embodiment 136. The method of embodiment 134, wherein the sequenceinformation comprises information related to at least 2 genomic regions.

Embodiment 137. The method of embodiment 134, wherein the obtainingsequence information of step (a) comprises using one or more adaptor(s).

Embodiment 138. The method of embodiment 137, wherein the one or moreadaptor(s) comprise a molecular barcode comprising a randomer sequence.

Embodiment 139. The method of embodiment 134, wherein diagnosing orprognosing the cancer has a sensitivity of at least about 50%.

Embodiment 140. The method of embodiment 134, wherein diagnosing orprognosing the cancer has a specificity of at least about 50%.

Embodiment 141. The method of any one of embodiments 134, wherein thenucleic acid is RNA, DNA, or a combination of RNA and DNA.

Embodiment 142. A method for preparing a nucleic acid molecule forsequencing comprising: obtaining a sample with cell-free nucleic acidfrom a subject, wherein the cell-free nucleic acid comprisesdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules; addinga poly tail to the nucleic acid comprising mixing the cell-free nucleicacid with at least one of a transferase or a polymerase, and at leastone nucleotide substrate; mixing one or more primer(s) under conditionssufficient to permit the primer to anneal to the poly tail of thenucleic acid; and mixing a modified reverse transcriptase underconditions sufficient to amplify the nucleic acid, wherein the modifiedreverse transcriptase comprises at least one improved enzyme propertyrelative to a wild type or unmodified reverse transcriptase.

Embodiment 143. The method of embodiment 142, wherein (b) comprises atransferase and a polymerase.

Embodiment 144. The method of embodiment 142, wherein the transferasecomprises a terminal deoxynucleotidyl transferase (TdT).

Embodiment 145. The method of embodiment 142, wherein the polymerasecomprises a poly A polymerase.

Embodiment 146. The method of embodiment 142, wherein the nucleotidesubstrate comprises at least one of dCTP, dGTP, dTTP, and ATP.

Embodiment 147. A method for preparing a nucleic acid molecule forsequencing comprising: mixing cell-free nucleic acid with anoligonucleotide-streptavidin conjugate, wherein theoligonucleotide-streptavidin conjugate is capable of annealing to thenucleic acid; and mixing the oligonucleotide-streptavidin conjugateannealed to the nucleic acid with a modified reverse transcriptase underconditions sufficient to permit transcription of the nucleic acid.

Embodiment 148. The method of embodiment 147, wherein theoligonucleotide-streptavidin conjugate comprises an oligonucleotideprimer.

Embodiment 149. The method of embodiment 147, wherein theoligonucleotide-streptavidin conjugate comprises an oligonucleotideprimer and an oligonucleotide acceptor.

Embodiment 150. The method of embodiment 149, wherein theoligonucleotide acceptor is in close proximity to the annealed nucleicacid so as to allow for template jumping.

Embodiment 151. The method of any one of embodiments 147-150, whereinthe oligonucleotide-streptavidin conjugate comprises a magnetic bead.

Embodiment 152. The method of embodiment 151, further comprisingenrichment of the annealed nucleic acid.

Embodiment 153. The method of any one of embodiments 142-152, whereinthe cell-free nucleic acid comprises at least one of ssDNA, dsDNA,dsRNA, ssRNA, extracellular RNA, and RNA from an exosome.

Embodiment 154. A method for preparing a nucleic acid molecule usingtemplate jumping comprising: (a) annealing a primer to a nucleic acidtemplate; and (b) mixing the template annealed to the primer with amodified reverse transcriptase, a polymerase with editing capabilities,and an acceptor nucleic acid molecule under conditions sufficient togenerate a nucleic acid molecule.

Embodiment 155. The method of embodiment 154, wherein steps (a) and (b)are performed sequentially.

Embodiment 156. The method of embodiment 154, wherein steps (a) and (b)are performed simultaneously.

Embodiment 157. The method of any one of embodiments 142-156, whereinthe reverse transcriptase is a DNA polymerase.

Embodiment 158. The method of any one of embodiments 142-156, whereinthe reverse transcriptase is a non-long terminal repeat (LTR)retrotransposon.

Embodiment 159. The method of any one of embodiments 142-156, whereinthe reverse transcriptase is an R2 reverse transcriptase.

Embodiment 160. The method of embodiment 158, wherein the non-LTRretrotransposon is an R2 non-LTR retrotransposon.

Embodiment 161. The method of any one of embodiments 154-160, whereinthe acceptor nucleic acid molecule comprises a protected 3′ end.

Embodiment 162. The method of embodiment 154, wherein the polymerasewith editing capabilities comprises at least one of 3′ to 5′exonuclease, T4 DNA polymerase, exonuclease I, Phi29, Pfu, Vent, KOD,exonuclease III, and exonuclease T.

Embodiment 163. A method for preparing a concatemer of nucleic acidmolecules for sequencing comprising: ligating a nucleic acid moleculewith a first adaptor; amplifying the ligated nucleic acid molecule byperforming a nucleic acid amplification reaction in the absence of aprimer to form a concatemer; and ligating the concatemer with a secondadaptor.

Embodiment 164. The method of embodiment 163, wherein the nucleic acidamplification reaction is polymerase chain reaction (PCR) or isothermalamplification.

Embodiment 165. The method of any one of embodiments 163-164, whereinthe first adaptor comprises a unique molecular identifier (UMI)sequence.

Embodiment 166. The method of any one of embodiments 163-164, whereinthe first adaptor serves as a primer.

Embodiment 167. The method of any one of embodiments 163-164, whereinthe first adaptor comprises single stranded nucleic acid.

Embodiment 168. The method of embodiment 167, wherein the singlestranded nucleic acid comprises single stranded DNA (ssDNA).

Embodiment 169. The method of any one of embodiments 163-164, whereinthe second adaptor comprises double stranded nucleic acid.

Embodiment 170. The method of embodiment 169, wherein the doublestranded nucleic acid comprises double stranded DNA (dsDNA).

Embodiment 171. The method of any one of embodiments 163-164, whereinthe first adaptor is different from the second adaptor.

Embodiment 172. The method of any one of embodiments 163-164, whereinthe first adaptor comprises two or more adaptors.

Embodiment 173. The method of any one of embodiments 163-164, whereinthe second adaptor comprises two or more adaptors.

Embodiment 174. The method of any one of embodiments 163, wherein bothends of the nucleic acid molecule comprise an adaptor.

Embodiment 175. The method of any one of embodiments 142-174, whereinthe nucleic acid molecule comprises at least one of ssDNA, dsDNA, dsRNA,ssRNA, extracellular RNA, and RNA from exosome.

Embodiment 176. The method of embodiment 163, wherein the adaptorcomprises a nucleotide modification.

Embodiment 177. The method of embodiment 176, wherein the nucleotidemodification comprises a methylated nucleotide.

Embodiment 178. The method of embodiment 176, wherein the nucleotidemodification comprises dUTP.

Embodiment 179. A method of depleting ribosomal and/or transfer RNA froma sample for library sequencing comprising: providing a samplecomprising RNA, wherein the RNA comprises ribosomal RNA (rRNA) and/ortransfer RNA (tRNA); performing an amplification reaction (e.g., apolymerase chain reaction (PCR) or an isothermal amplification) toconvert the rRNA and/or tRNA to double stranded DNA (dsDNA), wherein theamplification reaction is a partial amplification reaction or afull/complete amplification reaction; introducing a complex comprising anuclease or a polynucleotide encoding the nuclease and at least onespecifically designed guide oligonucleotide, wherein the at least oneguide oligonucleotide comprises at least one sequence complementary toat least one dsDNA and the nuclease or polynucleotide encoding thenuclease cleaves at least one strand of the dsDNA.

Embodiment 180. A method of depleting ribosomal and/or transfer RNA froma sample for library sequencing comprising: providing a samplecomprising RNA, wherein the RNA comprises ribosomal RNA (rRNA) and/ortransfer RNA (tRNA); performing an amplification reaction (e.g., apolymerase chain reaction (PCR) or an isothermal amplification), whereinthe amplification reaction is a partial amplification reaction or afull/complete amplification reaction to convert the rRNA and/or tRNA todouble stranded DNA (dsDNA); denaturing the dsDNA into single-strandedDNA (ssDNA) strands; introducing at least one specifically designedoligonucleotide comprising a binding molecule and at least one sequencecomplementary to at least one ssDNA strand to form a hybridized complexof the oligonucleotide and the at least one ssDNA strand; immobilizingthe hybridized complex to at least one solid support, thereby removingthe hybridized complex from the sample.

Embodiment 181. A method of depleting ribosomal and/or transfer RNA froma sample for library sequencing comprising: providing a samplecomprising RNA, wherein the RNA comprises ribosomal RNA (rRNA) and/ortransfer RNA (tRNA); introducing at least one specifically designedoligonucleotide comprising a binding molecule and at least one sequencecomplementary to at least one rRNA and/or tRNA to form a complexcomprising the oligonucleotide and the at least one rRNA and/or tRNA;immobilizing the complex to at least one solid support, thereby removingthe complex from the sample.

Embodiment 182. The method of any one of embodiments 181-182, whereinthe binding molecule is biotin.

Embodiment 183. The method of any one of embodiments 181-182, whereinthe at least one solid support is streptavidin.

Embodiment 184. A method of producing a cell free deoxyribonucleic acid(cfDNA) library comprising: providing a sample comprising cfDNA;denaturing the cfDNA to produce a single stranded DNA (ssDNA) sample;introducing, in the presence of nucleotides and/or a catalytic metal, acomplex comprising a template, a primer, and a reverse transcriptase tothe ssDNA sample, wherein the reverse transcriptase extends the primeron the template and subsequently template jumps to the ssDNA sample toproduce a double stranded DNA (dsDNA) sample (e.g., wherein the doublestranded DNA comprises a copy strand and an original strand (copy strandis the strand that resulted from the extension by the reversetranscriptase, and the original strand is from the sample)), and whereinthe dsDNA comprises at least one nick between the template and thessDNA; introducing a polymerase comprising a 3′-to-5′ exonucleaseactivity to generate a dsDNA with blunt ends and/or a 3′-overhang;introducing an (asymmetric) adapter comprising a nucleic acid duplexwith a single-stranded overhang at the 5′ end, wherein the (asymmetric)adapter is ligated to the 5′ end of the dsDNA, and wherein thesingle-stranded overhang comprises a sequence complementary to at leastone polymerase chain reaction (PCR) amplification primer; and performinga PCR reaction to amplify only one strand of the dsDNA (further whereinthe one strand of the dsDNA is the original DNA strand).

Embodiment 185. The method of embodiment 184, wherein the one strand ofthe dsDNA (original strand) improves fidelity of PCR amplification.

Embodiment 186. A method of producing a cell free deoxyribonucleic acid(cfDNA) library comprising: providing a sample comprising cfDNA;denaturing the cfDNA to produce a single stranded DNA (ssDNA) sample;introducing a terminal deoxynucleotidyl transferase (TdT) and adeoxyadenosine triphosphate (dATP) (and optionally a non-extendablenucleotide) to the ssDNA sample to generate a poly(A) and/or a poly(C)tail; annealing a complex comprising a primer and a first adapter to thetail of the ssDNA sample, wherein the complex comprises a sequencecomplementary to the tail; introducing, in the presence of nucleotidesand/or a catalytic metal, a reverse transcriptase and a complexcomprising an acceptor and a second adapter to produce a double strandedDNA (dsDNA) sample, wherein the nucleotides comprise degradablenucleotides, wherein the reverse transcriptase extends the primer andsubsequently template jumps to the complex to continue extension; andwherein the complex comprises a nucleotide block to prevent the reversetranscriptase from reaching the end of the complex and jumping toanother complex, wherein the dsDNA comprises an original strand and acopy strand, wherein the original strand comprises at least one nickbetween the complex and the ssDNA and the copy strand comprises at leastone degradable nucleotide; introducing a polymerase comprising a3′-to-5′ exonuclease activity to generate a dsDNA with blunt ends or a3′-overhang, and a DNA ligase to ligate the at least one nick;introducing at least one uracil-DNA glycosylase to degrade the at leastone degradable nucleotide to deplete/remove/degrade the copy strand(thereby resulting in only the original strand being present); and,performing a polymerase chain reaction (PCR) comprising at least a firstprimer and/or at least a second primer to amplify the original strand,wherein the first primer comprises a sequence complementary to the firstadapter and the second primer comprises a sequence complementary to thesecond adapter.

Embodiment 187. The method of embodiment 186, wherein the originalstrand improves fidelity of PCR amplification (i.e., removing the copystrand and leaving only or mostly the original strand improves fidelityduring PCR amplification).

Embodiment 188. A method for preparing a nucleic acid library forsequencing comprising: (a) obtaining a plurality of nucleic acidmolecules; (b) inducing a non-enzymatic intramoleculartransphosphorylation of at least one molecule in said plurality ofnucleic acid molecules by increasing a temperature of said plurality ofnucleic acid molecules, thereby providing a plurality of nucleic acidmolecules with a free 5′-phosphate moiety; (c) adding a phosphatase tosaid plurality of nucleic acid molecules with said free 5′-phosphatemoiety whereby the phosphatase converts one or more of said free5′-phosphate moieties to a hydroxyl group, thereby providing a pluralityof nucleic acid molecules with a free hydroxyl group; (d) mixing, in thepresence of an amount of adenosine triphosphates a product of step c)and a polymerase, whereby said polymerase generates a poly(A) tail fromat least one molecule of said plurality of nucleic acid molecules with afree hydroxyl group; (e) mixing, in the presence of nucleotides, (i) oneor more primers comprising a sequence complementary to said poly(A)tail; (ii) one or more acceptor nucleic acid molecules; and (iii) amodified reverse transcriptase, whereby said modified reversetranscriptase generates a plurality of continuous complementarydeoxyribonucleic acid molecule by reverse transcribing a sequence of anannealed template nucleic acid molecule, migrating to an acceptornucleic acid molecule, and reverse transcribing a sequence of saidacceptor nucleic acid molecule; (f) adding at least one solid support toa product of (e), whereby said solid support immobilizes an excess ofsaid one or more primers comprising a sequence complementary to saidpoly(A) tail; and (g) performing a polymerase chain reaction (PCR)reaction.

Embodiment 189. A method of producing a library for sequencingcomprising: providing a sample comprising ribonucleic acid (e.g.,cfRNA); subjecting the sample to high temperature sufficient to allowfor transphosphorylation of the RNA (optionally, further wherein acatalytic metal (magnesium) and/or a polyamine is introduced to thesample); introducing a phosphatase to convert a phosphate moiety of theRNA to a 3′-hydroxyl group; introducing an adenosine triphosphate and apolymerase to generate a poly(A) tail on the 3′-hydroxyl group of theRNA; introducing, in the presence of nucleotides, a primer, an acceptor,and a reverse transcriptase, wherein the primer comprises a sequencecomplementary to the poly(A) tail thereby annealing to the poly(A) tail,and wherein the reverse transcriptase extends the primer andsubsequently template jumps to the acceptor to continue extension;introducing at least one solid support to immobilize excess primer andnon-specific primer products to the at least one solid support, therebyremoving the excess primer and the non-specific primer products from thesample; and performing a polymerase chain reaction (PCR) reaction toamplify the RNA.

Sequences

AAB59214.1 reverse transcriptase-like protein [Bombyx mori]; SEQ ID NO: 1MMASTALSLMGRCNPDGCTRGKHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRFNQMTSVMGGGVGT18197 reverse transcriptase-like protein-silkworm; SEQ ID NO: 2MMASTALSLMGRCNPDGCTRGKHVTAAPMDGPRGPSSLAGTFGWGLAIPAGEPCGRVCSPATVGFFPVAKKSNKENRPEASGLPLESERTGDNPTVRGSAGADPVGQDAPGWTCQFCERTFSTNRGLGVHKRRAHPVETNTDAAPMMVKRRWHGEEIDLLARTEARLLAERGQCSGGDLFGALPGFGRTLEAIKGQRRREPYRALVQAHLARFGSQPGPSSGGCSAEPDFRRASGAEEAGEERCAEDAAAYDPSAVGQMSPDAARVLSELLEGAGRRRACRAMRPKTAGRRNDLHDDRTASAHKTSRQKRRAEYARVQELYKKCRSRAAAEVIDGACGGVGHSLEEMETYWRPILERVSDAPGPTPEALHALGRAEWHGGNRDYTQLWKPISVEEIKASRFDWRTSPGPDGIRSGQWRAVPVHLKAEMFNAWMARGEIPEILRQCRTVFVPKVERPGGPGEYRPISIASIPLRHFHSILARRLLACCPPDARQRGFICADGTLENSAVLDAVLGDSRKKLRECHVAVLDFAKAFDTVSHEALVELLRLRGMPEQFCGYIAHLYDTASTTLAVNNEMSSPVKVGRGVRQGDPLSPILFNVVMDLILASLPERVGYRLEMELVSALAYADDLVLLAGSKVGMQESISAVDCVGRQMGLRLNCRKSAVLSMIPDGHRKKHHYLTERTFNIGGKPLRQVSCVERWRYLGVDFEASGCVTLEHSISSALNNISRAPLKPQQRLEILRAHLIPRFQHGFVLGNISDDRLRMLDVQIRKAVGQWLRLPADVPKAYYHAAVQDGGLAIPSVRATIPDLIVRRFGGLDSSPWSVARAAAKSDKIRKKLRWAWKQLRRFSRVDSTTQRPSVRLFWREHLHASVDGRELRESTRTPTSTKWIRERCAQITGRDFVQFVHTHINALPSRIRGSRGRRGGGESSLTCRAGCKVRETTAHILQQCHRTHGGRILRHNKIVSFVAKAMEENKWTVELEPRLRTSVGLRKPDIIASRDGVGVIVDVQVVSGQRSLDELHREKRNKYGNHGELVELVAGRLGLPKAECVRATSCTISWRGVWSLTSYKELRSIIGLREPTLQIVPILALRGSHMNWTRFNQMTSVMGGGVGKMQ90176.1 reverse transcriptase [Lasius niger]; SEQ ID NO: 3MSGPGGGTPQHAGPSALSSKLDEFRLRVCSEGSALHGQDAAQRRAKRLATSPPHDPIDLELDPPPLSDAGLRGIKDELSAHDITFASINATSKESVNRKKELEEVIAAYRRAVDDLMMAYIKIKTERDTTAKIWKMMRSTSRGDSGSDLGIEIAGAVQESTGAAIRGMLGEWHARESERTKALVTEMTASMSGQFEGCVRRAVETLASFREAPSVPQIAGRSYAGAVRASGAVAGPQLPSGRRELRDQSRLETIEVVPGENMSKNLPDSEATCRAVLTSIKPSEAGIKVDRVIKGRNKTVRIVADQDEISRLRPMLDNLGMEVKRVDKLNPRLRIRDIPVGTDKSLFVKDLIKQNLDGASEEDIRLVYWSPAKGRMGPAAVIEVSPDIRIRLLNQGRVYLGWSSCRVADHLRVLQCFKCLGFGHTANNCQAGSDTCGHCDNVHNSVDNLNVGRLDGFVCPICLRSFSSKIGLGLHKKKKHPVEYNEEIVVARVRPRWTDEEIRLLAIDEAGAPPQTRSMNSYLLERRGDDRSLESIKGVRRKQAYKDLVAEYRGQLLDQRIDESLSQPDARPIAAMVDGVPLSGSVAAKDWLLAKCDSIIPEMNGGIWIRTAIRRLEEGQSPEGALDDWWNNVFSDLEVTGRKRVARGPRAIPVLSKRKARRIEYRRMQQLWRTNMTKAAHKVLDGDAGSLPHPTLAAQLGFWKPVLEAESVDLAWPFAVGHPGVAVGDLWSPITEGEVINIRLPRTSSPGLDGLTVHRWFTEVPAILRATILNIFMATGWVPPRFRHSRTVLIPKSSDLMDPAYYRPISVSSVILRHFHKILARRVAACELLDVRQRAFIAADGCAENVAVLSAILFDARTNRRQLHVITLDVRKAFDTVSHNAIRYVLSKHGMPQIMVEYLSTLYRTAAVRLEVDGEFSDEILPGRGVRQGDPLSPLLFNLIMNEILAEVPDQVGYCMMDRNVNALAFADDLVLIGATRDGAQRSLERVMAALYRFGLELAPAKCAAFSLVPCGKTKRIKILTDPQFVAGDRPIPQLGVLHTVRYLGVRFGETGPVIQGVELLPLLERITRAPLKPQQRLKILRTYLIPRYTHNLVLGRVSYSMLRKLDKQTRAAVRRWLVLPDDVPVAFFHCPIKQGGLGIQSFETAIPRLTLLRLNRLKDSQYEMARVVGSSAWADRRMRWCRFARRRDEDWPSELHAKVDGFELREAGNVSVSTRWLDDAMVHIPSSDWLQYVKVWINALPTRIRTTRGSRRLREDVNCRGGCGVQETAAHVVQQCFRTHGGRIMRHDAVASALAGELQRGGYNVHRERVFRTREGVRKPDILAAKGTHGHVLDVQIISGARPLSDGHDRKRSYYANNADLLARISALLQVPVRNLDVSTVTLSWRGVWARESAAVLTSLGVSKAVLRGITTRVLKGSYMNFSRFNQTTATCRGRANLRMSGWGPPKYB24671.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM-likeProtein [Tribolium castaneum]; SEQ ID NO: 4MSNPSSVPCLLASRGTVLLRGSGCARQGVRKSSSAAIQRALNVNVKKFKQARVNGGSNYDSLILLCYDFAAGRNADGEPAQNPCPYCARSFTTANGRGLHIRRAHPDEANNAIDIERIHARWSDEETAMMARLEAGAIQQGGVRFMNQFLVPRMPGRTLEAVKGKRRDATYKALVQRFLQAPQINLPELRDGDAPRQPDPQRENPPEPPAFDGAIRGAVADLVGGVDWQRLGFQGDRLCDIARRACDGEDVSGQLLGWLRDVFPVKRVSTRGDQSDLDVDGASVSRRTARRREYARVQELYRKDPKACLARILGDRREGANRAPNRDPAFIDFWRGVFSEASAEVEGWAEEVSDHGELARRVWDPISVEEVGRSRVRNGAAPGPDGIAVSVWNKLPPEAAALLFNVLLLGRCLPAELTRTRTVFIPKTDAPRTPADYRPISIASVVARHFHRVLSARVQRIPDLFTKYQRGFLSGVDGIADNLSVLDTMLTMSRRCCKHLHLAALDVSKAFDTVSHFAIVRACEQAGLPQPFVEYVRSIYGSAETVLEEGGRRHFVQVRRGVRQGDPLSPLLFNLVLDRALKRLSTDVGFRLTDATKVTALAFADDVVLCATTAKGLQTNLDVLEAELRLAGLLLNPNKCQALSLVASGRDHKVKLVTKPTFRVGQNTIHQVDASSIWKYLGIQFRGSGMCGCGSEGVAAGLKRITCAPLKPQQRMHLLRVFFLPKFYHAWTFGRLNAGVLRRLDVVVRTYVRTWLRFPHDIPVGYFHAPTKSGGLGIPQLSRFIPFLRLKRFDRLGRSAVDYVRECAFTDIADRKIRWCRERLSGIVDQVAGGRDALDAYWTAQLHQSVDGRALRESASVASSTQWLRCSTRAIPASDWLHYTAVHIGALPSRVRTSRGRRGGQDVSCRGGCLLDETPAHCIQVCHRTHGGRVLRHDAIAKRISVDLMELGWIVTREVSFRTTAGVFRPDMVAVKEGVTVILDVQIVSPAPTLDEAHRRKVAKYRDRADLARYLVEAAVARGRAPPANIRFASATISWRGVWSAESVGSLRELGLSARHFNRYTTMALCGSWRNWVRFNASTASRMGRGRGDASPRRHENQHDNDSLADLVRVALTKSDRGVLNDAVNRNLAQRAESLRIRKRGSKGKRKSKTGRHYGQTTSGSGQRAALFKKHQDLFLKNRRGLAETILSGKEDFGPRPEPPVTSVEEFYGGIFESPSSPDNKPFERQIPVAAVKTMSELELAILFNIILFRNVQPSAWGVLRTTLVPITISSALQRLLHRVLAARLSKLISLSSSQRGFTEIDGTLANALILHEYLQYRRQTGRTYHVVSLDVRKAFDTVSHCSVSRALGCFGIPSVIREYILATFGAQTTIKCGSVTTRPIRMLRGVRQGDPLSLVLFNFVMDELLEKVNEKYEGGSLQSGERCAIMAFADDLILIADRDQDVPAMFDDVSTFLERRGMSVNPAKCRALIAGAMHLSIWGKMQ90064.1 reverse transcriptase [Lasius niger]; SEQ ID NO: 5MNVGEDVAVLDVGRPNSHVCPICLRSFTSRIGLGLHKKKRHPVEYNEEICIARVKPRWSEEEIRILAMEEARAPPRTKSMNVYLWERGDESRSLESIKGVRRKQSYKDLVISYKDQLLAEKVDESLERQCSVPVVPAETPLPGSEAAMKWLVARVDDIIPEMNGGIWVRAAVCRLREGCLPDAALDDWWRNVFSDLETARSGRISRGPRVAPVLSKRRARRVEYRRMQQLWKVNMTKAAHKVLDGDTDSLPHPTLAEQLDFWRPVLEASSASFVRPVGAGKVVDGLESVWSPITEGEVINIRLPPSSAPGLDGLTVNRWFAEVPAILRATILNIFMATGRVPPRFSGSRTVLIPKSLDLMDPSCYRPISVSSVVLRHFHKILARRLAAFNLLDTRQRAFIAADGCAENIAMLSALLFDARANLRQLHVLTLDVRKAFDTVSHDAIRYVLRRNGIPAGMVEYLSTLCRTSTIRLEVGGAFSDELFPGRGVRQGDPLSPLIFNLVMNEILAVVPEQVGYNMLGHNINALAFADDLVLVAATREGAQRSLDRVVAALSDFGLELAPAKCAAFSLVPSGKLKKMKVLSDPQFAAGGCAVPQLGVLQTMRYLGVWFADAGPVDREVELLPLLDRITRAPLKPQQRLKILKTFLIPRFIHILVLGRTSYGLLRKLDRQIRAAVRRWLRLPEDIPKAFFHSPIASGGLGILSYETAIPRLVLARLDRLDKSQYDAARMVGSSAWAVRKRRWCGLAKRVEENWPAEFYGMVDGFELREAGNVTASTNWLDDPMIRVPSSEWLEYVKVWINALPTRIRTTRGSRRLREDVACRGGCGVQETAAHVIQQCFRTHGGRIMRHDAVASTLAGELQRGGYKVRREHVFRTPVGVRKPDILASKGERGYVLDVQIISGARPLTEGHKRKRNYYAGNAELLAKARESATTLTSLGVSKAVLRGITTRVLKGSFMNFARGPALTRQASVTSAEASSAHICDFPGCGRTFSTKTGRGVHQRRAHPDWFDGQQTTAMVKARWSEEETLLLARKEVELVRQGERFINQALFEVFPERSLESIKSKRKQPAYRDAVGTIMDSIAREDNAGVPLNVPVPDSANLKRNIEEHLSALPAPSSSAFMSARLAGICDSLTRKSQVAVVEELSLYLRSVFPIKPRGARPSGNVVTDAPSKRQERRAEYARAQDLWRKNRCKCLRMLLDDITGVNVPPKETMVPFWETIMTGNFPTSPGCDVLAPATNDLWLPITALEIRRALPAGTTSAGPDGLTARFLRRVPMEILERILNVILWCEKAPTHLMESATTLIPKKSNAHTPSDFRPITVSSVLLRTLHKVLATRMARLVQIDQRQRAFRPTDGCSENVFLLDLILRYHHRHHKPLFMASLDIAKAFDSVSHKTIEETLAIMGIPSPMRAYIMDVYQRSSTVLCCGSWTSRKIQPTCGVKQGDPMSPIIFNMIMDRMLKQLPGDIGTRIGGSTINAAAFADDMLLFASTPLGLQKLLDKSTDFLRKCGLQVNTSKYMTISLRNVPREKKTVVDRETVFLCQDKVLPALKRTDEWNYLGIPFTPKGRMKLNIAQKLQSSLDKLTKTPLKPQQRLFGLRVMVIPGLFHQAELGNINISVLRKCDRLVRCRVRQWLSLPSDVPNAYIHANVKDGGLGITALRWTHTGRIKRHDAIVSFVSRLLEVQGYDVSVEPRIKSDHGLKKPDIVAKLGVTAIVLDAQVVNDQISLDEAHQRKIDYYQDIEGNVKETFRVQNVIYSSITLSWRGLWSQKSVNSLTDLGIIKKKHIKIISTRAIIGGLTSFHIFNKATYVQGRAGACJ71597.1 reverse transcriptase [Rhynchosciara americana]; SEQ ID NO: 6MSNYNETNTSGGDNPRMATQTTGSLSSGPINQHTCELCCRTFGTRAGLGQHVRKTHPIESNQSINVERKKRRWSPEEIRRMANMEAQATINNIKHLTQYLATYLPQRTLNAIKGRRRDAEYKELVTGIIANLRSNSSTQQTNQVCNESEMSQRSKILQSIRESVRDLRSRRNKYAKALQELGEAALCGKMLNEEQLIHCIKSMFNTAKCPKGPRFRKTATHSGTNKQQRQQRYARVQKLYKMNRKVAAKMVLEETDKIQIKLPDHDPMFKFWESEFKEGEGMPERMPKDLKESPDLKAIWDPVTEEEVRKAKVANNTAAGPDGIQPKSWNRISLKYKTLIYNLLLYYEKVPHKLKVSRTVFIPKKKDGSSDPGEFRPLTICSVVLRGFNKILVQRLVSLYKYDERQTAYLPIDGVGTNIHVLAAILNDSNTKLSELHVALLDITKAFNRLHHTSIIKSLVGKGFPYGFITFIRRMYTGLQTMMQFEGHCKMTQVNRGVYQGDPLSGPIFLLAIEKGLQALDKEVGYDIGDVRVNAGAYADDTDLVAGTRLGLQDNINRFSSTIKQVGLEVNPRKSMTLSLVPSGKEKKMKVETGKPFRANDVPLKELSINDFWRYLGISYTNEGPERLSLTIEQDLERLTKAPLKPQQRIHMLNAYVIPKYQDKLVLSKTTAKGLKRTDRQIRQYVRRWLKLPHDVPIAYLHAPVKSGGLNIPCLQYWIPLLRVNRVNKITESQRSVLAAVGKTALLTSTVYKCNQSLATLGGNPTMLAYRTYWEKELYAKVDGKDLQNARDDKASTRWNGMLHSDISGEDYLNYHKLRTNSVPTKVRTARGRPQKETSCRGGCKSTETLQHVVQQCHRTHGGRTLRHDRIVGLLQHELRRDYNVLAKQELKTGIGLRKPDLVLIKDDTAHIVDVQVARCSKLNESHVRKRSKYDKKEIEVEVKSRYRVSKVMYEACTISYKGIWDKQSVMSMRRLGVSEYCLFKIVTSTLRGTWLCWKRFNMITSVRSAFM44926.1 R2 protein [Eyprepocnemis plorans]; SEQ ID NO: 7MVGPTTRSKKGTGPPTGASVPTTESASQSSGAGRALLPASCPLCQRSFTTVNGLGQHRRRAHPEVVNAEIQTDRKKARWSKEELMRLAHAEARLVIEGCRFLNQELLKSFPDRTLDSIKGQRKGKAYRESVASFVSQLRSSVASAGPSGASPVAEEPLPGSPSVSQDDEVDAAIWSALADLPSSGSRFKLVDDIVALGPNTSRDIVRSMLPSALDAVGSKEPAAHPPLPFGARRPPDKKRARRRWEYAAVQRAFRKNAARCVNGLLDGTLLHQPPSIPGLVEFWKDLFTAPCASSRPRSKEGLSPMLLASSQPVSFRDLWAPITSEEAAAALPPRNSAAGPDALTPAQLRRLPHPVFLKILNLFLLARSLPSRLLRARTTLLPKKTSPASPADFRPITVCSVLARAFHKVLAGRLMRYCVLDGRQRAFIPQDGMLHNSFLLDLAMAHSRRTACSLYVASLDVSKAFDSLDHGALSPVLRAHGLPVEFVEYVRGCYQASTTVICGGGSSSDLVRPSKGVRQGDPLSPILFNLSIDLLLSRLPGYIGARIFSRRVNAAAFADDILLFAETKGGLQELLSTATSALGDLGLEVNPFKCFSLALVASGREKKVKVDNSVIFRAGNKNIPALAMGDTFRYLGLQFSTSGLSQFHPRQEVQEQLDIIKRAPLKPQQRLFALRSVILPGTYHGLALGRTRLGALKSLDVCVRAAVRAWLRLPDDTPIGYFHAPVIYGGLGIPATRWLGPLLRRRRLASMEGLGVIVDEPSQDILKREICRLDNYLKWDGDVIKTSYQLGRFWALRLHSSVDGAALRRSAQTPGQHSWVSNTRLMLSGRDFLACVRARISALPSRARLLRGREGDTRCRAGCNASETNNHVIQHCWRSHEARVERHDAVALYMVRGLRRRGYDVHRELHLRTSQGLKKPDIVAVSGTTAFVIDAQVIGDHLDADRCHREKVEVYDQQPVHTEIKRMFPEVQMITTTSATLNWRGVWSPASAKALIGIGFNSNHLSTMATRALLGSIMAARRFDSMTAPRRRMMPRTGVGAHN53448.1 reverse transcriptase, partial [Nuttalliella namaqua]; SEQ ID NO: 8QTIRGLGQHIRIKHPTVSNAKISVATSKVRWTDEEIHLLAKEEARLIKLGKKYLNQELQSYMPHRSLEAIKGQRKSEIYKERVKHLVETKLLEVEPTMSEVLEPTESQKKDSFQKEIEKIITNTPPRKFQGERLWEIARKAIRGEDIYRDLNDYVVDTFVVATKPHPQAKGKSYTTAPPSSRRKRRRRLYGRMQEMMRRKPADCAKIVLDGERVASKINSEEFFDYWEKLTTKEPSNWPLSSDTAQRENLKDPMFPVTLREIKENLPSPHSAPGPDGCSARLLRAVPPLTLQLVLDLLLFVRRPLASLKGARTIFIAKKDAASHPKDFRPISISPILLRFLHRILARRLNRMVPIDKVQRGFQPRDGCAECAILLTMAIKESRSKLKSLYLASVDISKAFDSVTFEALDSALKRVGLSEGFIGYIRDLYTSGNTLFQFDNQCRTVVPTNGVRQGDPLSPFLFNIVLDEFFSTMEQEIVFDKNGLNLSALAYADDLVVFASSQRGLQHRLEEAYSFFKKKGLEINVEKSFTLSLQAAGKEKKIKVREDMRFNVAGMVLPALDINSVFGYLGVSFSPLGRPSETWEELKDYLDRISSAPLKPQQRLYILRGFLVPRLIYRLVLGRWTAGTLLKLDRQIRAAIRRWIGLPHDCPLGYFHAKIGSGGLGIPSMRTMIPELLLRRLTKLEMNETMGTKEIPKCESYWYYVKKMEDLTKYDGHKLDTKYACQRYWAQKLYKSVDGRDLEASSKVPFANHWILDHTRWLPGYEYCKMVKFKVNAMPVLTRTSRGLDRPRNCRGGCDQQESLKHVVQHCHRTHGARIKRHDNIVDFIGKRLAAKGYGVLKEPRVKTSTGILKPDLVVTGDDRVLIIDAQVVGSGQYLTEDHLRKVKKYNIDEVKDYFKEPRKCVAVTSVTVSFRGVWCANSAKELIELGLTKNDIKVVSAICLQGGVRAHSLFSNVTSVLKHRACJ46647.1 reverse transcriptase [Triops cancriformis]; SEQ ID NO: 9MSQKRRPEKAVPDEGATAHDVAQPDKSKCSVCGETFKGPASVTMHMVKKHPVEFNELKMAKKPVPKKVRWSEEEIFQLARTEAELTLQGVRFINVELQKIFPAREIEGIKGQRKLAKYKELVKDQLDEIGRAPNPPEQEIGEDVPSPFKAWLELLLALPKTPNDFLEHKLDNIIVQALKEDVNSDQVFNDLNSYLKLILEPSGRAKSVPGEIIHGDPSGSAKTSVTKAPKPATVSSSRKKRRDAEFARIQRLYRKNRTSCINTILDGNTREHEAPKNMEGFWREIFERESPDDPDDPDIFLEEEASDIWKYISFYEMCNLYPPPSTAPGPDGFSSKDLRRMTPRVLNKILNLLLHLRDLPQILKSHRTVLIPKTDLPTKPGDFRPITISNILVRHLNKILANRVSHLIPINERQKAFLPIDGCAENIFTLDFILHHARTKIKSLSMAILDISKAFDSVSHEISIFRALREARCPIGFIKFIENCYGGCFTKLFCGGVKYPSEVSMNRGVKQGDPLSPVLFNLVIDGLIRQIPSALGFNVSDQVKVSCIAYADDLILIATTRAGLKTLLDLTNSYLAKRGLSLNPDKCSALSIVASGKQKLVYIASSEHFDLAGQKMRNLNVGDSWRYLGIQFSHLGRAEKVTPDLTCLINRLQKAPLKLQQKLYALRIYLIPRLIHGLTLSKTNLGELKTLDKLIRKYIRAWLHLPDDTPMGYFYTPLKAGGLGLPSLRLVILNNRLERILRMKASQDIIVRTIAESETLGVEIRKLHDLLSIDGTILDTSVKIHSFWAERLYSSYDGKCLCNSANFPPGNKWIGEDSLNQRSHIFADCLKLRINALPTRSRTARGRPLKDKPCRAGCRNSDGVKVIETLNHITQVCERTHGARVKRHDRLVDFAVKGLQRPHRVVLKEPHYKTVNGVRKPDIVIKIPDHTYICDFQVVSDTSCLELEFRKKALKYAEDKGLCDQLTRDHPGELSFTAITFNTRGLIAKSSVTALRKLGMPPRSIMTLQKICMEGSLEIWRIFNQTTAMARNBAC82590.1 reverse transcriptase [Ciona intestinalis]; SEQ ID NO: 10MGEWPWVSWSLTVLVEKWRPFTILQPYPMPGQLRVDVYLPRKTSYLMDKNIYENTTSPGGGPLCGEKTHRSDVIIPPPGFAPSTDTASNTLGENVDASATTSSANPLSQEPGWCESCSKLFKSQRGLRVHQRSKHPELYHSQNQPLPRSKARWSDEEMVIFAREEIANRKIRFINQHLHKVFPHRTLESIKGLRGKNVRYARIMADLEAEMTSQPEAATSLCTETSENLASSNVLPQTRGWAENLVENIDTAHLANLGPLSQFEPGKPSSSTKEAINTEYNDWISKWLPSGAAHRERRANPPSTKLNARATRRLQYSRIQNLYKLNRSACAQEVLSGAWKVQSGELNLKEVQPFWEKMFRKESAKDRRKPKPTGEVLWGLMEPLTIAEVGSTLKSTTPSAPGPDKLTLDGVKRIPIAELVSHYNLWLYAGYQPEGLREGITTLIPKIKGTRDPAKLRPITVSSFICRIFHRCLAQRMETSLPLGERQKAFRKVDGICHNIWSLRSLIHNSKDNLKELNITFLDVRKAFDSISHKSLGIAAARLGLPPPLITYISNLYPNCSTKLKVNGKISKPIEVRRGVRQGDPLSPLLFNAVMDWALSELDPRVGVQIGEQRINHLAFADDIILVSSTKIGMVSSINTLSRHLAKSGLEISAGKEGKSASMAIVVDGKKKMWTVDPLPRFKVNSQKIPALSITQQYKYLGINIDAQGARNDAARILTEGLAELSRAPLKPQQRLYLLRVHLLPKLQHGLVLSSCAKRALTYLDKSVRSAIRRWLTLPKDTPTAFYHAKACDGGLGITRLEHTIPILKRNRMMKLTLSEDPVIMELVKLTYFTNLLHKYSNVKLLNSWPVTDKDSLARAEASMLHTSVDGRGLSNCSDVPRQSDWVTNGASLLSGRDFIGAIKVRGNLLPTKVSAARGRQREITCDCCRRPESLGHILQTCPRTWGPRISRHDSLLKRVRNQACLKNWTPIIEPSIPTNIGLRRPDLVLAKGNIAFLVDATVVADNANMQLQHEAKVEKYNNSDIKEWIKVHCPGVDEVRVTSLTANWRGCLYGGSASFLTEDLGLPKAELSLLSAKINEKGYYLWCAHYRGTARLWNRPLRSAAB94032.1 reverse transcriptase domain protein, partial [Drosphila mercatorum]; SEQ ID NO: 11FERRTGPVGYLPSGIEKLMVQMFNNEPRIAENNSVEYTPTVTRLGDHQVRNADANLQTMFPCRECERSFRTKIGLGVHMRHRHKDELDTARRRVDVKARWNEEELSMMARKELELTANGERFINKKLAEIFTNRSVDAIKKCRQRDNYKAKIEQLQGQAALISEANEPPTTQRRPSLSELEVTPSSSHSVPIAPPPIHSDDILLQELQGMSPVAVRRSWRVEVLQSIIDRAHISGKEATLQCLSNYLLEIFPNRNDRPSSATVPARRPRNRRISRRQQYARCIKSLLDGTDESALPNQSIMEPYWRQVMTQPSPSLCSNTVPRKGNMQEGVWSPITSRDLQVHKVPLTSSPGPDGITSQTARSIPIGIMLRIVNLILWCGDLPVPFRMARTIFIPKTVRANRPQDFRPISVPSIVVRQLNAILASRLTAAVSWDPRQRGFLPTDGCADNATIVDLVLRDFIHKRYASCYIATLDVSKAFDSVAHDAVFNTVTAYGAPKSFVDYVRRWYSGGGTYFNGGDWRSEEFVPARGVKQGDPLSPVLFNLIIDRLLRSLPKDIGVHVGNAKVNACAFADDLMLFASTPKGLQELLNTTVKFLSSVGLTLNADKCFTISIKGQPKQKVTVVEQRTFCIGRARVQLKRSEEWKYLGIHFTADGRARYNPSEDIGPKLERLMQSPLKPQQKLFALRTVLVPQLYHKLTLGSVALGVLRKCDKLVRSFARKLLGLPLDVSVAFYHAPHSCGGLGIPSVRWIAPMLRTKRLAGINWPHLEQSEVASAFLSEELRRARDRAKAGVNELLSQPKIDTYWADRLYTSVDGNGLREARRYAPQHGWVSQPTRLMSGKAYRTGIQLRINALPTRSRTTRGRHEMNRQCRAGCDAPSHNHVLQRCHRTHGSRVSRHNGVVSYLKKGLETRGYTVYSEQSLHGQNRVYKPDIVAFRHDSTIVVDAQVVTDGLDLDRAHQSKVEIYNRQDLLTTLRSVYRARENIEVVSATLNWRGIWSFQSITRLRTLGILTAGDSNVISSRVVSGRVYSFKTFMFHAGFHRGMAAAC34906.1 reverse transcriptase, partial [Forficula auricularia]; SEQ ID NO: 12LGPRSINQPRIRTDSPNIVRPSGSTTTMQRCTTSDLTFSQCRFPHCKFRRPSLTGVRVHEQRSHKAFFDRLQAEVIRNQTSKKKPRWTEEEKNLLALAQANLIIEEETNIIDDLVSKFTYRTKDALKSQIRKPEHKTRVSEFTLAIQAHIDNIMLPAPVPIATDPLQVSCNFKDRAKDYIDTLEPITSTKFYLDELEALCDNICIWPTRLLITTVESYIRKLFKTTSTLKPAQKLHNPSNRNLPKRQLRRMEYGKTQKLWKKNPCRAIKTIIDDKDCKSPPEREAMTQYWKTTFSSKKRTCPQYEPRESTKTQLWEPVTIEELHCCHLEMTTSPGPDGITVRQLYLVPEQLLVRILNLLMACGKMPDSFLESKTTLIPKKPNSTEPGDFRPITVQSVLVRQLNKILAARVAQHIPLDERQRGFRPVDGVAHNIFELDMILRCHRSEFRDLRLASLDIAKAFDSITHNTIEDTMEVRGFPKPMINYIMACYRRSKTRFTFNGWISDTVKPTCGVKQGDPLSPILFNLVMDRMIRKLPKEVGVNVGSKHYNGLTFADDLLLFATTPEGLQSSIDIVHLFLLECGLLINKQKSFVLTVKAYPKLKKTAVIVTEKYMLDRHILPAIDREKLFHYLGVPFTAEGRCRDDTIAHLKRKIDVLTKAPLKPQQRLFALRVVILPSCYHILTLGGSNLSLLKKIDLMVRAAGRKWCCLPKDTPNAYFHASSRDGGLGLPSMRWLIPLHRYLRLLRYEGRNPEDTNVYLTTEINRAKIRLSDNGSNIDCQAKLWQFWADRLYKSVDGSALIESSKVPQQHRWATGGSRFLTGRDFINSIKLRINTLPTLSRTLRGREGNRMCRGGCYNVETLFIHVLQVCHRTNGTRVKRHNAIRQYIARGAAVKFDTVEREPRIKSASGAVNIPDLVACNSDEVVVIDTQWWDQANLDEAHQAKAEKYAHLSDILKHKYSRDRVKFTSVTLSFRGLWSKQSLKELTDLGIVNSKDIQIISTRAIIGGIASFRMFNSTTSVNSVNSFLEIALGBAC82589.1 reverse transcriptase [Ciona intestinalis]; SEQ ID NO: 13MPGCQSVVGSECSQCGRVFKTARGLSVHRRSKHPEAYHREHIAAPRVKARWSEEEMILMAQXEVQAPAGTRFINQYLHALFPSRSLDAIKGARGKSAAYKKIILEQRLAALVPVSPPNQISGAESPSQPSNTSQEVRQDPEERSLRLRDAIDLGHLRTDFDMTAVNPGHPDPVIREEIDREFLRWLEPNRRRRGYAKATRIALNAPGKVRRRAEYAAMQRQWKTKRGLCAREALEGTWKIPARTVSLSDQEAFWRPLMESQSKNDLREPAKVGETLWGLLDPITPDEVRQILGSMSSKAPGPDGHRLSDLRSIPIDQICSHFNLWLLAGYQPKALRMGESCLIPKVKDASRPQQFRPITLGSYVGRCLHKCLASRFERDLPISIRQKAFRCMDGVAENVMILRSVLDDHKKRLAELNLVFLDVSKAFDSVSHRSILHAVKRLGVPPPLLKYVEELYADSETFLRGSGELSPSIKVRRGVKQGEPLSPHLFNAVIDWALSSLDQSFGVTVGEARVNHLAFADDIVLLSSSQPGLQRLIDQLTTHLGESGLRVNSTKSASIRIAVDGKNKRWVVDPRDSVHVGGVRIPAVAVSGSYRYLGVNISAAGMRVDAADSLASKLANLSRAPLKPQQRLYILCTHLLPSIYHQLVLSSTSKKFLKYLDRCVRVAVRRWLRLPKDTPKAYFHAKCNDGGLGVPELQRVIPLQKAGRWLKMTRSQDPVVQAAVGLEYFQKLLERWSTPELYQWGGGGITTSGHLAVAQARSLYSSVNGRGLRQSGLVSTQFDWVRSGCSLLSGRNFIGAMQLRGNLLATKLRASRGRPRVDISCDCCRTPESSGHILQVCPRTSWGARIGRHDNVAKLVARESAKRHWKVIREPAIPTPAGIRRPDLVFSKGDTAIVVDVTIVPDNAELSDAHSSKVSYYDNGAIRGWVALNTGASHITFSSVNNNWSDCMAEESKRMLKLGLGLPNSIRGTISAVVLEKGFHMYLCFKRGTFRAS YAIL01110.1 reverse transcriptase [Bacillus rossius]; SEQ ID NO: 14MLASSFKKKPRMVSSSKSGSSCNDAPTGVVVPASKESVESPSLDKKVGYGCEFPGCPRVFTTKTGRGVHHRKAHEDWYDARQKLDYVKARWTREESALMAREEAKAGTQSAKKMNQVLQLVLPDRTLESIKSHRRSAQYKELVLQAMGALSDSGKCAGPSQLANAELSTPLSSAVLGXGEPGGGGSGEHGSGDVPGSSRGEHLSALLDGLQPGPVVDRLRLIVRSVDDWSRARLHQEIGWFLRDLFWKPLTPNLARVSLPSKDKVSRRRLRRADYGRVQRAWKRNRNTCLRDLLRDKRTESAPPEELXVPYWESVLRSGSSCTPGQRGRTAERTELWDPVSSKEVEQALPPLGTAPGPDSFTPKDFRAVPSAWACIFNIFMLCGRLPDYLLESRTTLIPKRDGACNPEDFRPITVSSVVVRCFHKVIANRMSRHIQLDPRQKAFRSLDGCSEGVFLLDFILGHARRNHRPVHLASLDVAKAFDSVSHAAILDVLRSFGVPDQMVEYIASVYAGSRTRLQGDGWQSHAIHPTCGVKQGDPLSPMIFNMVIDRLFTLFPRDTGVSVGDTVLNGMGYADDLVLFATTPVGLQQLLDITAEYLSQCGLRVNAAKCFSVSLAIVPHEKKVVVATKHRFKCLGQPIPALKRSDQWKYLGVPFSPEGRLKIDPLGRLKDELEKLRRAPLKPQQRLYALRTVVVPGLYHLLVLGGTTISSLNRLDIAVRSTVRKWLSLPHDVPNAYIHADARDGGLSIPSYRWTVPRLRFHRLKALSVLCDGGGPDEMVACVGDEIKRASARLQDHGMNINTRNTYRVRFARLLHTSNDGAPLKGSKKVEGQHRWVTDGSLMLSGRDYIACNWVRINSIPLRKRTARGRVRDTRCRAGCDSTETLFIHVLQQCHRTHDMRIKRHNACVKYLLDRQRSRGKTVFWEPHFHTAGGLLKPDSVILHDASTAVVVDALVAGERSDLDREHDRKVSKYEPLVDLVKDRYSVDKVIFSSLIISARGVWGGRSFRHLSKLRLLDISDAKVLSTRVLLGGMGAVRVFNRRTAVSGRVNGWAFO19998.1 R2 protein [Lepidurus couesii]; SEQ ID NO: 15MSGKSSKPRTVSSGSSSQETPPSGSNACDICGKCFMKPVGLSLHMSKVHPTQYHARLEKNQPKAKKFRWTDEDLYFLAKKEAELLLLGGIKFMNKELAEFFPEKSVDQIKGQRRSETYKQQVVSIHSELLKLQAVADSPPPSRIPAKEVSAWLDFLLALPKTKNKFSEDKLDQLIRTAQEGTPVLNDLDLYLREVLVQPTRQGERQAKPLPPPKSSREKRDREYARVQNFYRKNKTACVNAILDGNKKCENKIPDIDEFWKAIFESQSPPDAEPVSYVVDEEPKNIWSWISFFEMNRNYPDTSTSPGPDGVTARMLRSIPARVLNKLLNLLLFIEDLPAVFKCHRTVLIPKVDNPALPGEFRPITISSIIVRQLNKIIAARVSEGVPINPRQKAFRQIDGCAENVFLLDFILRDAKTKIKSLSLATVDIKKAFDSVSHEISIFRAIRGARCPENLVNYIQNSYSGCTTQISVGGSISTTKILMNRGVKQGDPLSPVLFNLVINEIIRKLPASIGYPINSELSINCIAYADDLILVANTREGLKLLLNLLNEELPKRGLELNASKCFGLSLTALGKLKKTHLCTSDQLDLHGTLIKNLTAEESWVYLGVPFSHIGRSKSFSPDLEALLNKLQKSPLKLQQKLFALRVYLIPRLLHGLVLSRVAIGELKIMDKLILKHLRVWLRLPKDTPLGFFYSPVKLGGLGIKNLRTNVLKCRKQRIERMLVSPDDVVRLVAESEIFLKETDKLKDLLTINGMCLDXRNVPRTGKNNKFWSERLYTSFDGKTLAYSEYFTQGGGWIREDKILQPAHVFAECIKLRINALPTKSRVAHGRPTKDRSCRAGCLDVQKVPAIETINHIAQVCPRTHGARIKRHDRLVQFLSLNLRKNPKRNVLVEYNFRTVAGIRKPDIIVIEDTRAAILDVQVVGDSSNLEMEYLEKSRKYSNDATLSMRINALQKLYPTVTSLTFHAVTFNNRGLIAKSTVAALRMLGVPPRCIMILCVISLEKTLEVWRMFNQSTASARK KMQ88340.1 reverse transcriptase [Lasius niger]; SEQ ID NO: 16MGGSPPASFRRRPGKEGIPAPPGPWGCQPLGLVLVGPKNRNQASDNSGGPSGPSAPANPPSQVDDADFRCEFPGCNRTFPTNRGRGVHHQRAHKDWFDARLQPAVDKVRWTAEETAMLARKEAELTVESNPRFINQELLQYFPQRTLEAIKGKRRNQEYRELVEEFVEEFRNPDVITIEDDEEDEEDQRDIFLDYLESLTRPQGREFQATRLHNIAMEARTSGKDATLQKIALYLREVFPAPPPRRERRRKKTPNPAPMRKREARRCEYGAAQSLWKRDRRHCITNILNEMGPVNQPPRETMEPYWTRMMTTDGRTSPPSDKVPIKEDIWTPITGNDIKRSRIPRASAPGPDGISARLYRSIPTTVIIRLFNLLLWCERLPEDLLLSRTIFLPKKTNASEPGDFRPITIPPVLVRGLHKILAKRLETALDIDPRQRAFRSMDGCADNTLLLDTLLRYHRKQYKSLYMASIDVSKAFDAVTHPTIESTLISLGVPPPMIRYLGQVYANSRTRIEGDGWTSKPVHPKRGVRQGDPLSPILFNAVTHRLLQRLPREVGARLGNIPINAAAYADDLLLFASTSMGLQQMIDTMTDYLAECGMTINVEKSMTVAIRAAPHLKKTAVDASLSFSCGGRQLPSLKRTNKWRYLGVVFTPEGRAQCRPAEVVAPLLGALTKAPLKPQQRLYALRTVVIPKLYHQLALGAVTIGTLNKTDRLVRGALRKWLALPHDTPNAYFHTSVRDGGLGIPAIRWTAPVQRRGRLLGVMKALGQQGLDRFIQDELNTCKKRLTDHGVLLGTPEMVAKRWAQQLYGSIDGAGLKDSAKTPHQHQWIADGSKFLTGKDFINCNRARIGALPTRSRTTRGRPQDRRCRGGCLAQETLNHVLQHCHRTHGQRIKRHDAVVKYIARNMPRSGYEVHQEPHYKTELGLRKPDLVAVLGQTAIIIDAQVVSEQTNLDDAHTRKVAYYNEPATIRAIKAEHGVRTVKVTSATLSWKGVWSPRSAEELRKLGFIRAGDAKVVATRVLIGNIAAFRTFNATTSVEHRAGIGAF019997.1 R2 protein [Lepidurus couesii]; SEQ ID NO: 17MSEESRPKQTASKRGAAVEKTMMSGTYVCTLCGRSFEKSVGLSLHTNRMHPEAYNKLKEAKKPVLKKARWSEEEVFLLAQKEAELSFIGGIKFMNIELHKIFPERELEGIKGQRKNPTYKAQVVSLLAEIRESKANDSSSSSSSSSSCDSASLGISNWLEFLLALPKTSNQFQEGRLDRLISDALRGVDVLENLDAYLLEVFAKPMAQNPCPKPPPPAKNSRERRDREYSRVQNFYKKNRSACINSILDGNTRSQNVIPGLTKFWTETFEKNSPPDDEAPDQFVADEPRDMYKWITFYEMSQDYLDSSTAPGVDGFSAKQLRSMSPRVLNKILNLLLLSENLPNSFKMHKTVLIPKIDDPKSPGDFRPITISPVLARLLNKILAARLSKLVPISQRQKAFLPVDGCGENIFLLDYILRSSKKSSKSVAMAVLDVKKAFDSVSHEISILRALNEAKCPINFINFVRNSYDGCTTKLTCGGTSFPDSVRMNRGVKQGDPLSPVLFNLIIDSAIRKLPDSIGYVIRDGLKINCLAYADDLILVASSRAGLKTLLNIVAEHLSLRGLDLNAAKCHGLSIIASGKAKTTYVSAADSLDLDGQPIKNLGVLDTWTYLGIPFSHLGRAEKVSPDLTNLLNKLQKAPLKLQQKLYAVRNFVIPRALHGLILSKTNLKELNTLDRAIRVFLRTLLYLPKDTPLGFFHSPIKSGGLGITCFRTSVLKCRLQRIARMRSSCDGVIQAVAESDIFADEYAKLRDLIRINGNVLDTTESIKRYWAQRLHSSVDGKTLAYMDYFPQGNLWMSEDKVSQRSYVFADCVKLRINAIPTRVRVSRGRPNKEMCCRAKCFDSQRMPAFESLNHITQVCPRTHGSRIQRHDKIAKFLFKNLNNCPSRSVLYEPHFVTVDGLRKPDIIIYDDSHMVVLDVQVVSDSANLEKEFECKAKKYANDVALRSAMLIKYPFIKSFSFVAATYNNRGLIAKSSVQVLRQLGLSPRSIMVSILTCLEGTLETWRIFNQSTMNAHAAC34903.1 reverse transcriptase, partial [Anurida maritima]; SEQ ID NO: 18VGVQGEVTSLRLLCQQDSEVYATTMNRKNNYTRGNRFSSGSPGNFVVVRDPAQDLPFKCAFCERTFTTSNGKGLHELRSHPKEYNMRVPVAKKRARWSEEELSQLAEAEHDLKSKKQYASELDLSRDLEGCMVGRSLESIRGQRKLPRYVEIFNRLSNSRASIPEIGEGDEEANSVGSDEVFHASGHGTGITEALEVLVSKRPGEAFREEVLNGIVRAKLEGSEVFVRLEQYLSRMFVGQSSLASTPCSSGEGKLPLTCSGSNKKEQRSDLFSRFPEKGPSVSVSAPSVSDMGRKRRKALSRNEATQVREEFVEFARNVDPIPRRKCARVNEGLQGNQRLPKEKPMTARAKMRHLRLLRYRRLQELYKKDRSLAAKQVLQDMLDSKPGRNPEAVKYWAETMGKESTGIDVSVMTGRPRYRDNVWSPIYPGEVSAAVKLMDSSGATGPDGFSVRSLKCTPSRVLAKVFNLFLLEEKLPAFLMTSRTVLVPKVKEPKAPTDYRPISVSSTLVRLFHKILARRLTLASGLDSRQRGFVPVDGCAENLVVLESAIRSAKNYKRSLFVASMDIKNAFGSVAHEAIFEALSKSGAPDSFVTYVRNCYDGFASVVKLGRDTAQTTVRQGVLQGDPLSPILFNLVIDQIIRSLPETVGVQLDANTKLNSMAFADDLILLSSSEAGMRRMLGVLAGVSSKFGLIFHPGKCKYLAMIWAGKQKKMKIATDLSFEIGGGFMTPVGVTETWKYLGAYLGQIGIQPARLSLQTFLERIAKSPLKPQQKLYLIRVHLLPKLIYPLVMAPIRASMLNKLDRMVRVALTGKDGILHLPQSVPSAFFYAPIGEGGLGLMELRTSIPAMVKARFERMMNSTCHHVRAAAKGAANSNRIALANRFLRKTADGIPVTSAKLVKEYQAAKLHGSFDGKPLSEAGRVKGIHSWTCDGRMVMTGQAFCEALKIRINALPCLSRYNRGTEKPRECRAGCKTTESLNHVLQVCPRTHDMRVARHDKLVNRLGGYLSQKGFEIHTEPRIITSLGLRKPDIIAIKGEKGVVLDAQIGGAANLNAAHDAKMCYYSSSPEIKEWVTGKGAPDVSYGACIVSPQGIMSEESWKTLRGLGFSKGMLNSLVVTVMEQSTYVWHVFNRSTASYGWKRRRKRKWDAF019995.1 R2 protein [Lepidurus apus lubbocki]; SEQ ID NO: 19MSEESRPKQTASKSGAAVEKTMMSGTYVCTLCGRRFEKSVGLTLHTNRMHPEAYNKLKEAKKPVLKKARWSEEEVFLLAQKEAELSFIGGIKFMNIELHKIFPERELEGIKGQRKNPTYKAQVVSLFAEIRESKANDSSSSVSSSSSVDSASMGISNWLEFLLALPKTSNQFQEGRLDRLITDALQGDDVLLNLDAYLLEVFAKPMAPNPCPKPLPPVKNSREKRDREYSRVQNFYKKNRSACINSILDGNTRSQNVIPDLMKFWTETFEKVSPADDKAPDQFVVDEPRDMYKWITFYEMSQNYLDPSTAPGVDGFSAKQLRSMTPRVLNKILNLLLLCENLPNSFKMHKTVLIPKIVDPKSPGDFRPITISPVLARHLNKILAARLSKLVPISQRQKAFLPVBGCGENIFLLDYILXNSKKKSKSVVMAVLDVKXAFDSVSHHSILRALNEAKCPVNLXNFVRNSYDGCTTKLTCGGTSSPDSVRMNRGVKQXXPLSPVLFNLIIDSAIRKLPDSIXYLIRDGLKINCLAYADDLILVASTRAGLKTLLNIVAEHLSLRGLDLNAAKCHGLSIIASGKAKTTYVXALESLDLDGQPIKNLRVLDTWTYLGIPFSHLGRAXKFSPDLSNLLNKLQKAPLKIQQKLYAVRNFIIPRALHGLILSRTNLKELNTLDRAIRVFIRTLLHLPKDTPLGFFXAPIKTGGLGITCFRTTVLKCRLQRIARMRNCSDEVLEAXAESDTFDDXYAKLRDLIRIXGNVXDTTENIKKYWAQRLHSSVDGKTLAYMDYFXQGNLWMSEDKVSQRSYIFADCVKLRINAIPTRVRVSRGRPNKEMSCRAKCLDAQRIPAFESLNHITQVCPRTHGSRXQRHDRXAKFLFKNLNNCXSRKILYEPHFMTANGLRKIDIIIYDDSHLVVLDVQVVSESANLEKEFTCKAEKYANDVALRSAILLKIXIYBAC82591.1 reverse transcriptase, partial [Ciona intestinalis]; SEQ ID NO: 20KFKQSKVKHNQKENKSKRQIRRAQYARTQINYKRNRQQAINSILSKQWRELDDRGPPLEHVLPFWQKLLEKSSEHDTRQPEPVSGPIWELVEPIVAEEVRLVRKSMPTSAPGPDGIRHKDFMAVQPQILADNFNLWLLTGYQPKSCRLGRTILIPKEPGTRDPAKHRPITINSLLIRCFHKILSNRIMKLIPLSPQQKAFRPGDGIAEHIWNLRHILNQHKLEKKELNLAFLDVRKAFDSVNHSTLLLAAGRLGVPPPLLKYIENLYEGSSTTIIANGERSPRIRVRRGIKQGDPLSIPLFLGVMDWAMSNLNPNVGSSIGGKSINCMAFADDLVLISRTQIGLQTNLDTISYNLNQSGMTLNSEKCATLRLAVDGKSKKWWIDHRPFLRVEGAKCGAMDIEGTYKYLGVRVGAGDTRAECKEKLMSDLKETTEAPLKPQQRIFILRNYILPRSLHILTFTNTTARLLKQLDSAIRIHVRRWLKLPKDTPLGYLYSDYKDGGLGVPRLLSRVPLLRIRRMAKYNTSEDPTTVALRSCHTFTSAAVKWAMPPKISNVVISDKRKERDFHRSELATSIDGSGLSCANTTPRAHQWVVNGTGLLSGKNYIGAINARGNLLHTASRAARGRPQRTTDCDSCHRVETLSHILQSCPRTHGPRIRRHDKVTKVIAEAAGKKGWKTIAEPRIPTPEGIRKPDLILYQHGRAIVMDTTIVSDSADLSMSHQHKVIYYQNECIRSWVQEATGATEVTWSSFSANWRGCIADESRDLLLHDSTSQKTAACAX83712.1 endonuclease-reverse transcriptase [Schistosoma japonicum]; SEQ ID NO: 21MSAMLNITTECHPVSTGVNADPSFSYSSTCLCCFSSFASNSLLLDHATASHSANIVSPPSESGSFQLVCMLCSSHYLSSRGFTQHLRHVHPDAYNSLKCLRLNASPLTHRSWSSEEDVCLLSRADELSASCRLKVDLYNRLHTVFPSRSPEAIKKRLRFLSSSSESSSSAPSPSDISSAASSVSSLPVDLDAPTRSLIPTRRPARSHSSSILSFFTRTPRDVSSSASPVVLHSSPSTTIVINNASVRLHLDSPVSSHALESSCMSSLATNPLHIIDNQHLAASPPLSGVHCPSVPSSAPAAAHVDQSFDNPALGSTPVDSCPPVTSLTLLPAQLSDVAVNLMTSCPELIKPASPSPPLMDDNLLQLILPSDSPNMSPHIVPPPDVVVAPMCTDSTRELISAALRLITQNSPLMHAPTLREFLQAALVNMPDMIEIQLFLNSHAELQFPTKWRPSKPRLPPTYRANTSRKHLRRLQYGHIQTLYNRCRRDAANTVLDGRWRSPHTSSPFSIPEFETFWKTIFTTPSTPDNRPVVPVLPTCPALLDPITPDEITWALKDMRNSAPGVDRLSAQHFLNFDVPSLAGYLNMVLAFKFLPTNLSISRVTFIPKGASPQQPNDFRPISIAPVITRCLHKILAKRWMPLFPSSKLQFAFLQRDGCFEAINLLHSLLRHAHERHSGCSIALLDISRAFDSVSHHSILRAAHRFGAPDGLCQYLQRVYNGSTSLFNTVDCAPSRGVKQGDPLSPLLFIMSLDEALESIETVSPVIVDGLPISYIAYADDLVILAPNADLLQKKLDKLASLLQRSGLIINTSKSMSIDLIAGGHSKLTALKPTVFKIDGNQLQRLNVSDHFDFLGISFDYKGRSKMDHVETLSAYLLNLTQAPLKPQQRMSILRENLEPRLLYPLTIGVVHKCTLRQMDCLIRSSVRKWLRLPSDTPTSFFHSSISTGGLGIPHLSSIIPLHRRKRAAKLLLSPCPIIRWVSQSPSFSNFLRICNLPINVHRDLIHSFDEARCSWSKQLHSTCDGRGLSMSSRNTVSHLWLRYPEHIFPRLYINAIKLRGGLLSTKVRRSRGRQENADLLCRGRCGHHESIQHILQHCSLTHDIRCRRHNDICRLVASRLRRNNIRFFQEPCIPTPVSFCKPDFIIIRDSIAYVLDVSVCDDANVHLSRQLKINKYGCSTVVSSIYNFLNATGLRISSVRQTPLIITYRGLIDPLSTTSLRRLSFSSRDISDLCVASIQGSMRIYNTYMRGTSPQDPXP_009165216.1 hypothetical protein T265_13057, partial [Opisthorchis viverrini]; SEQ ID NO: 22VLVTFDNVYYESSGASAPVPTSTQERLVGFTCEECGKCCKSKAGFVAHHRVHDNESVGTNTVAQLACADCSRLFPTKIGLSQHRRHAHPTQHNADKLGRVKYSGTRWSQQESQSLLRLANNLYPSCGTQTELFTRLEQYFPGLSAISIETRLRVLNWQAQQDESSSGEPERIIGLTTADSSEADGYNVWFKQTVDCTVSLLESHAHRALASVDLLAFARGLQSGIMTPEQVLSLLDLHAFRTFPHTWKTVSRRRRQLAHRIPVNRKQIRRANFAAIQTLFHQRRKDSASAVLDGSWRDLYKGNCGLPTNAERPWKQVLSAPKRAGSRPSRIVVPSDWSLVEPITGEEVGRTIRLMGNSSPGLDKLTPRTLRRFNANVLAGYFNLFLLSGGCPPHLCRARITLVPKVPHPTSPDQLRPISVSSILVRCFHKFAFLHRDGCLEATSLLHALLRHSSATASNLSLAFVDISKAFDSVSHDTIVRSAKAFGAPSPLVRYIAQSYENAVAVFPSSEVHCHRGVRQGDPLSPLLFIMAMDEVLGLSMPQLGYQFHDTLVDGFAFADDWVVCAESQARLKEKLEAAAVELGRAGMKINARKTKVMVICGDRKHRATAVSVEPFRFAEELITPLGPTDTVTYLGIPFTSKGKGVFNHRQHLLKLLEEVTRAPLKPHQRMDITRNYLIPRLTYSLVLGQVHRNTLKRLDNYIRQYIRGWLRLPKDTPISYIHAGKQHGGLGIPSLSATIPMQRKTRMEKLLSTQCRVLRNVVNDSAFGKIVRDLSLPIRVHGACVNTKEELVAAWGESLHNSADGRGLRELVTSPLSNRWLVFPERVFPRIFIRGIQLRCNLLRTRVRSARHGHGGQTILCRGNCGQPESLVHILQSCWITHDARCARHNRVARELAKRLRRLGYTVFEELRAPTSTSFIKPDLIAVRDRRATVIDVSIVSDGRGVIVWNEKKQKYGADEHSLAIISALHAIGCDINFSVHQPMIISYRGICFPQSAKAVIGLGLLRSDVSLFSPIFPRYSFTFKTRIHFLSQVGKTTLILSLVSEEFSPKVPAQAEEITIPADVTPERVPTQIVDYSSRTQSHEQLCAEIRRADVICLVHALDDEKSLERISSYWLPLIRHNGANPDCHSPIVLVGNKLDLLNESKLSKALPIMSEFSEVETCIECSAKTLLNLSETFWFAQKAVLYPTAPLYDAERKELTPACIRALTRVFRICDTDNDGYLSDRELEAFQSRCFSVPLTTQSLQDVKQLVRQSCPGGVTLNGITQKGFLFLHLIFVQKGRHETTWTVLRQFGYDNQIRLSNEFLFPRFSVPSGCSTELSTLGIQFLHMLFNKYDLDRDGCLSPSELSEMLAIFPEDQLSHVSELTDSVTTNSTGWITCQGFLAYWALTAYLEPTRVLEYFAHLGFTYFAAGSFWSTVNSHHQQQQHPKDPYDGTPSTPLLIGALPLRDRLIGQNPPNSSGRSDAGGSSVPMSRNTRDALLRSLVITSEKRLDTIRRSTQRTVFYCRVYGARKVGKTCLLQGLLGRHLRGTGGLAIGGLSGRSSGWAAATGIQVYGQQRTLIMHEIGAAGGEQXXXXGGPRRIQVYGQQRTLIMHEIGAAGGEQMTAGEALSADVACLVYDVSDSDSFRYVANLFLNYYRGTRVPCLFVESKSDQPRVVQNYQVDPIELTTKYNLNPPEPFSSMNLEQCLNALASNSTRNVASSLGDPVSRSRSATNLFPNPMPSRRAGSLEPSSSTAGGETHLLKDSSHKLSPGIGLSLLSLEKESVQLNPPLDARGRRHSKPDLAYRPSMEFSARDTNFLPVYVTLCTLANYPHLRGLQLAQTDYAWKWTLAATILAGFGFVAFRIAKTHFAAB94040.1 reverse transcriptase, partial [Hippodamia convergens]; SEQ ID NO: 23AFADDVILCGTTSWGLQRNLEIFEEELRRSGLSLNPGKSKCVSLVASGREKKVKLVMTPTFRASGSWLSQVDGTTFWKYLGLQFRGCGMAGCGSDDVAECLERLTRAPLKPQQRMHLLRVFLLPRFYHVWTFGRLNAGILRRLDIRVRNAIRTSVRLPHDVPVGYFHAPTNAGGLGIPQLSRFIPLLRLKRFERLAHSSVESVRECARTEPAVAKVRWCRERLADVVDRVADGTQSLREFWTRELYRSMDGRALRESVRETPSTQWLRCCTRVIPARDWLNYISVHINALPSRVRTSRGRRDGVDVTCRGGCLTAETPAHCIQVCHRTHGGRVLRHDAIAKALSVHLTQRGWSVRREVSYQTVVGVRRPDIVLLAGREIAVVDVQVVAPNPSLDSAHRKKVAKYRDEAQLATCLVRGASVQPRRRAEEETQVRFASATISWRGVWSSESARSLRELGLTDRELAQYSTYDLRGSWMNWVRFGASTSTRMAWRPAAV85443.1 reverse transcriptase-like protein, partial [Amblyomma americanum]; SEQ ID NO: 24PLFFNLVIDEFLEGLNHELAYRCQGLQVSAMAFADDLILAASTKDGLKEHTRKLENFLSQRGLRANAEKSSTLVILPSGRGHKSKICPNITFQIHGKDMTSQNCTSLWRYLGVTFSATGRIQGPIRFELAALLQRVSSAPLKPQQRLVVLRYYLMPRLTHRLTLGPISAKTLTAIDRTIRSNIRRWLALPLDVPMGFFYAQIEQGGLGINCLRTTIPSLRLRRFAKMTHSSNSACTFAATRRTVTDSIHQAERLCVFKGQTLRNPKESGRFWSSQLHASSDGRSLQGCKNAKGSTYWLREGTSFLKGREFIDLTKFHTGAMPNLTRLKRGRDVPKKCRAGCESEESMGHIQQRCHRTHHTRIERRNNLVKYLSKRLHDLGWHVKVEPHYATSQGTRIPDLVIKRDSQALILDVQVVGTRVGLTQAHEAKTNKYKIPELLMSMDPRPSVSSVTTSYRGVWATQSVGILTDIGLGIHDFKIMTIRCLQGGLRSFRTHQQMTSVRRDGNFRAVAAV85445.1 reverse transcriptase-like protein, partial [Ixodes scapularis]; SEQ ID NO: 25PLLFNLVVDEFLQTLDPGIGYSSDQLQLDGMAFADDLIVFASTPEGLQRRLDSLHSFVGARGLAINAEKSFTVSIVPSGREKRTKVVTTGNFSVSGIPLPACGIEARWKYLGVEFSPNGRNVPLTKDVASMLERVAKAPLKPQQRLVILRFYLIPRLYHRLVLGRWNRKLLKRLDVQIRDAVRKWMALPHDTPLGYYHAPVAEGGLGVGSFSTAIPWMQVQRLPRMATSSSPICRQAADTYMVKTALDRAKKACVVRGNILSDKQSVGKHWSALLHASNDGHALREVGRSPAAQRWVQEGTGLLTGRAFIDVNKLRINALPVRTRTKRGRDTDKNCRGGCRAPETLDHVLQKCHRTHAARIKRHDGLVQIVVDRLRKKGWTVEVEKRFNGPQTLIPDIVARRVLRENTPLEKRESAVIDATVITCGYPLLKAHESKVRKYDVPQVTSICKGKNTEHPLVTSATLNFRGVWCKQSAQDLLSLGLTKQDLKIMTVRCLQGGIHCFRVHHGMTSVKKRY44798.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Trichinella britovi]; SEQ ID NO: 26MAHWYASLIRLIAAGTFIGERPANSENSQMPCHLISDATCGFSFATFSGLHLHRKRAHPDVFAAACGKKTKVRWSNDEISLLATLEAGLDPACKNINQVLAERLMEYDITRGVEMIKGQRRKEQYKALVRQLRSKSVTQQCVGFAGSMDSNVPVNDTTSSVASEVTITYPEYGAVMSCDLIKEATGMAMVDINELQSNIRKVFSSGRKLPVKSRGARETVQKKMANPRVAKYKRFQRLFRSNRRKLASHIFDKASLEQFGGSIDEASDHLEKFLSRPRLESDSYSVINGNKSIGVAHPILAEEVELELKASRPTAVGPDGIALEDIKKLNSYDLASLFNLWLKAGDLPESVKASRTIFLPKSDGTTDISNCRPITIASALYRLFSKIITRRLAARLELNVRQKAFRPEMNGVFENSAILYALIKDAKARSKEICITTLDLAKAFDTVPHSRIVRALRKNNVDPESVDLISKMLTGTTYAEIKGLQGKPITIRNGVRQGDPLSPLLFSLFIDEIIGRLQACGPAYDFHGEKICILAFADDLTLVADNAAGMKILLKAACDFLEESGMSLNAEKCRTLCISRSPRSRKTFVNPAAKFNISDWKTGISSEIPSLCATDTFRFLGHTFDGEGKIHIDMEEIRSMLKSVRSAPLKPEQKVALIRSHLLPRLQFLFSTAEADSRKAWLIDSIIRGCVKEILHSVKAGMCTEIFYIPSRDGGLGLTSLGEFSLFSRQKALAKMAGSSDPLSKRVAEFFMERWNIARDPKVTEAARRVYQKKRYQRFFQTYQSGGWNEFSGNTIGNAWLTNGRARGRNYVMAVKFRSNTAATRAENLRGRPGMKECRFCKSATETLAHICQKCPANHGLVIQRHNAVVSFLGEVARKEGYQVMIEPKVSTPVGALKPDLLLIKADTAFIVDVGIAWEGGRPLKLVNKMKCDKYKIAIPAILETFHVGHAETYGVILGSRGCWLKSNDKALASIGLNITRKMKEHLSWLTFENTIRIYNSFMKNAAV85444.1 reverse transcriptase-like protein, partial [Rhipicephalus microplus]; SEQ ID NO: 27PLLFNLVIDEFLAELDPQLAFTSEGMKVSAMAFADDIILTTATHWGLKQQIDRLNSFLGARGLKINAAKSTTLVIEPSGWQKRSKIRTDIDFFVNGERLATTNCTSTWRYLGVHFGVKGLEKGLVRRQLAILLERVSKAPLKPQQRLVVLRFYLLPRLYHRLVLGPILAKTLLTIDRVVRSAVRRWLALPLDAPLGFFYAAVEEGGLGVPCFRTVVPAMRLRRYQTVAQSSNPACAFAATRPTITQLKRQADNLTIFKGTKIGNSKQSRKYWARQLHMSFDGRPLQQCKEAPGSTSWLGNGTSLLRGREFIDLAKFHVAAVPNLTRLRRGRELPKQCRAGCQAEESLGHILQRCHRTHHARIKRHDNILRYLAGRLTELGWQVQQERHFKTSQGTKIPDLVIIKREKSHILDVQVVSTRVELTEAHHHKCDKYKIPDLLFQVSPTPTVSSVTLSYRGTWAGESVRTLQEVGLTRNDFKMMTIRCLQGGLAAFKMHQMSTAVVRRGVGSQGKRX52183.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM [Trichinella sp. T9]; SEQ ID NO: 28MSNRLANTAAAGGVPEKNSGTLDIPGQPSSSGEKRAISYPGPFGCNSCSFTSTTWLSVELHFKSVHNTREFVFLCSKCEKSWPSINSVASHYPRCKGSVKAAVVPSSLANTCTTCGSSFGTFSGLQLHRKRAHPDVFAASCSKKTKARWSNDEFILLARLEAGLDPACKNINQVLAERLMGFNITRGVEMIKGQRRKDQYKALVRQLRSNSETQQCVGLAGSMDSNVPVNDTTSSVASEVTITYPEYGAVMSCDLIKEATGMATVDINELQSNIRKVFSSGRKLPMKVRGVRETVQKKMANPRVAKFKRFQRLFRSNRRKLASHIFDKASLEQFGGSIDEASDHLEKFLSRPRLESDSYSVISGDKSIGVAHPILAEDVELELKATRPTAVGPDGIGLEDIKKLNTYDLASLFNIWLKAGDLPDSVKASRTIFLPKSDGTTDISNCRPITIASALYRLFSKIITRRLAARLELNVRQKAFRPEMNGVFENSAILYALIKDAKVRSKEICITTLDLAKAFDTVPHSRILRALRKNNVDPESVDLISKMLTGTTYAEIKGLQGKPIIIRNGVRQGDPLSPLLFSLFIDEIIGRLQACGPAYDFHGEKICILAFADDLTLVADSAAGMKILLKAACDFLEESGMSLNAEKCRTLCISRSPRSRKTFVNPAAKFNISDWKTGVSSEIPSLCATDTFRFLGHTFDGEGKIHIDTEEIRSMLKSVKSAPLKPEQKVALIRSHLLPRLQFLFSTAEADSRKAWLIDSIIRGCVKEILHSVKAGMCTDIFYIPSRDGGLGFTSLGEFSLFSRQKALAKMAGSSDPLSKLVAEFFIERWNIARDPKVIEAARRVYQKKRYQRFFQTYQSGGWSEFSGNTIGNAWLTNGRARGRNFIMAVKFRSNTAATRAENLRGRPGMKECRFCKSAIETLAHICQKCPANHGLVIQRHDAVVTFLGEVARKEGYQVMIEPKVSTPVGALKPDLLLIKADTAFIVDVGIAWEGGRPLKLVNKMKCDKYKVAIPAILETFHVGHAETYGVILGSRGCWLKSNDKALASIGLNITRRMKEHLSWLTFENTIRIYNSFMKNKRX72028.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Trichinella sp. T6]; SEQ ID NO: 29MSNRLADTDAAGGVPEKNSGALDIPGQPSSSGEKRAISYPGPFSCNLCSFTSTTWLSMELHFKSVHNIREFVFLCSKCEKSKPSINSVASHYPRCKGSVKAAVVPSSLANTCTTCGSSFGTFGGLQLHRKRAHPDVFAASCSKKTKARWSNDEFTLLATLEAGLDSACKNINQVLVERLMGYNITRSVEMIKGQRRKEQYKALVRQLRSKSETQQCVGFAGSMDSNVPVNVTTPSVVAEVAITYPEYGAVMSCDLIKQATGMAIVDINELQNNLRKVFSSGRNLPAKSHGVRESVPKKMANPRVAKFKRFQRLFRSNRRKLASHIFDKASLEQFGGSIDEASDHLEKFLSHPRLESDLYSAISGDNSIGIAHPILAEEVELELKATRPTAVGPDGIALEDIKRLNPYDIASLFNLWLKAGDLPDSVKASRTIFLPKSDGTTDISNCRPITIASALYRLFSKIITRRLAAKLELNVRQKAFQPEMNGVFENTAILYALIKDAKVRSKEICITTLDLAKAFDTVPHSRILRALKKNNVDPESVDLISKMLTGTTYAEIKGLKGKPITIRNGVRQGDPLSPLLFSLFIDEIIGRLQACGPAYDFHGEKICILAFADDLTLVADSAAGMKILLKAACDFLAESGMSLNTEKCRTLCISRSPRSRKTFVNPAAKFIISDWKTGVSSEIPSLCATDTFRFLGHTFDGEGKIHIDLEEIRSMVKSVKSAPLKPEQKVALIRSHLLPRLQFLFSTAEVDSRRAWLIDSIIRGCVKEILHSVKAGMCTDFFYIPSRDGGMGLTSLGEFSLFGRQKALAKMAGSSDPLSKRVAEFFIERWNIARDPKVIEAARRVYQKKRYQRFFRTYQSGGWNEFSGNTIGNAWLTNGRARGRNFIMAVKFRSNTAATRAENLRGRLGMKECRFCKSATETLAHICQRCPANHGLVIQRHNAVVTFLGEMARKEGYQVMIEPKVSTPVGALKPDLLLIKADSAFIVDVGIAWEGGRPLKLVSKMKCDKYKIAIPAILETFHVGHAETYGVILGSRGCWLKSNDKALASIGLNITRKMKEHLSWLTFENTIRINNSQMPRHLISDAYEWINKIPSVPIYYLAKPQPRERAWQNQRGKKTLLSLTLVAFO19999.1 R2 protein, partial [Lepidurus couesii]; SEQ ID NO: 30KQGDPLSPVLFNLVINEIIRKLPASIGFPINDKLSINCIAYADDLILVANTREGLKRLLEILNEELPKRGLELNASKCFGLSLTALGKMKKTYLCSSSHLDLHGTLIKNLTADESWVYLGVPFSHIGRSKSFSPDLEALLNKLQKSPLKLQQKLFALRVYLIPRVLHGLVLSKVAMGELKIMDKLILKYLRLWLRLPKDTPLGFFYASVKLGGLGIKNLRTNVLKCRKQRIERMLVSPDDVVRLAAESEIFLKETVKLKDLLTYDEKCLDTTEKINKFWSERLYTSFDGKPLAYSEYFPQGCGWIREDKIAQPAHIFAECIKLRINALPTRSRLARGRPTKDRSCRAGCLDVQKEPAIESLNHIAQVCPRTHGARIKRHDRLVQFLCLNLKKNPKRNILVEYNFRTAAGIRKPDIIIIEDTCAIILDVQVVGDSSNLEHEYLEKSRKYSNDANFINAFQKLYPLVTNLSFHAVTLNNRGLIAKSTVTALRRLGVPPRCIMILCVISLEKTLEIWRIFNQSTAAARKAAA21258.1 reverse transcriptase, partial [Drosphila ambigua]; SEQ ID NO: 31PLGSQGDPLSPIIINMIIDRLLRVLPNEIGATVGNAITNAERFADDLVLFAETPMGLQKLLDTTVDFLSSVGLTLNSDKCFTIGIKGQPKQKCTVVIPQSFCIGSRPCPALKRSDEWKYLGIHFTAEGRTRYSPAEDLGPKLLRLTRSPLKPQQKLFALRTVLIPQLYHKLTLGSVMIGVLRKCDIVVRSFIRKWLGLPMDVSTAFFHAPHTCGGLGVPSVRYLAPMLRMKRLSGIKWPHLVQSEAASSFLEVELNKARGRTLAGENELTSRTAIETYWADKLYMSVDGSGLREARLFRPQHGWVFQPTRLLTGKDYRNGIKLRINALPSRSRTTRGRHDLARQCRAGCDAPETNNHILQNCYRTHGKRVARHNCVVNNLKRILEEKGHTVHVEPNLQGESAVSKPDLVAIRQNHAFVIDAQIVTDGLSLDQAHLPKVERYKRPDVITAVRRDFNVSGAVEVLSATLNWRGIWSNQSVKGLITNNLLTTSDSNVISARVVIGGLYCFRQFMYLAGYSRNWTKRX12851.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Trichinella nelsoni]; SEQ ID NO: 32MAAIRVNYPGPFTCQKCTFTETVFARFVTHCTHHALNVNLACSICGKDFTSINAVASHFPHCKKGTKRNETPIDAPTNTEMHVSTHNTHICTVCSRAFNSFAGLRLHEKRAHPATFAASSQKPTKHQWTIDHLREAKEVEDQLTDSNSCSAKSFAEALSSKWSEAISVDMAKYLRKKLRRVDINFNAHNRGTTDGDTSGLLPEVVGKSISLEGVGGKNRAALIGEEIHGVGVLSMRKTSGFRVEIVGQTTPSPNKSSNALSPQHRDRDPGRDLRHHLEDSLTSCNSELEGLLNFLTNKILNSKPEDRNKYVDDVISIVTEFVCKPNGHQPPPAPKKRTKEEPKNRRQKIRSKYAKMQSLFKRDPKRIAAHLIKNQPLCNVSCPIDAAESALRQRLSQRPSVDAAPFTSKCPPNSKNILDPISPEEVTLHLQRMKIHTSAGPDGIQVSHLRSCDPVCLAKAFNLFLLARHIPQQLKDCRTTLIPKIDHPRPDAEDYRPITVASSLYRLFSKIVTRRLEDSLSLHPRQKAFRSGTDGAFDNTSTLMTIIRNAHKRGRELNIVSIDLAKAFDTINHTSIDRALRMQGLDVDSRALIAQMVTGSSTIIKGDGGAFSNRIEINQGVRQGDPISPLLFNSVMDELIERLEKSKVGFKFMGEEITTLAFADDVTLISRSHRGMAKLLSITLDFLNERGLSLNVNKCKGIRLVRTPKTKSLVQDTSKAFRVPNIGEEKQYIPMVPSGNVIKFLGIQITLNGKPHFELAPLEGTLERIRKAPLKPAQKLATVRDYLIPSLEYKLGVPGISMKLLESVDAAIRLTVKRFLHLQITGMNSMFLTMPIKEGGLGLRPLTTQRIARVAVGTNSMMTSMDNVSRVVANTATLRKPLLSALEHFAVPAATKGAIREGKQNLLREEIAQLSETYQGSCLPSFKKGSLVNSWLRGTCGMRSRDYITGLKLRFGVIETRSQKWRGRTPQNPDALLCRHCGHLSGYRETAAHISQKCPTTHATIIQRHNKIVNLVADRAKREGFAVHVEPAIKSGGNVFKPDLVLVKDGAAHIVDVAVPWEKGTSMHEKHQKKTNEYSPTVEEVRAQFDAETCTVGAIIIGARSSWCPSNNRSLKACGLHLPKKFKRLLCRVALEGTCKIFQNFF TLTKFD59471.1 hypothetical protein M514_11684 [Trichuris suis]; SEQ ID NO: 33MVKSLPGKGLELLFNLWLYLGDVPSRLKECRTVLVPKRYPPQGPGDYRPITIASTLYRVFTKILANRLSQCVELNRRQNGFMKGINGCGENTFILSTALESSRRHARELNLASLDLQKAFDTVSHASIRRALCRQNVPTKGIQLLMNLLSNSYTRLDHANGMSAPIPMCRGVKQGDPLSPLLFNLVMDELLDELEGAGGGFTFSPSTQVNCLAYADDILLLSDSRAGLQSNLLRCSRFLSARSLRLNIQKCSTLRMYKVPRIRSICATRDPMFYLDPTDESTLLPAFTGAEFLHYLGVDFNPYGRRRDQVAGALVLLARVRRAPLKPQQKIELIRNHLLPRLLYSLTVGNPLANTGRTIDKKIRQCVKEILHLPASTMCDDFFYVPRSRGGLGFLNLQEATDLSVLRLMVKMRTNDDDVARTSAEQWFNQRRFAKLAARRGLPTANLGALHKVKEEIAKKHQERFQRSYQGSGHAEFHDKRSNAWMGGEQMTGRGYINAIKVRASLVPTRVQTLRGRADPGDHRTLCRRCGDVSRAPESLAHISQTCAFCHGLIIRRHNAIVQKLAQLAQTQGFQCTTEPIIRINDQTHKPDLVLAKNSSCWTIDVSIPWESRDPLDRRHMQKCQKYRCLAEPVKKLTNSTEFSTGAIVIGARGAWCERNDDTLSRAGLSITDRMKQLLCLITLEKTCQLISWFMRSTDRLALRQPTRHRSSAQRTEARIPRSVDQAAFGSPARHHVAASPRHAPENRTVPAAHKXZ75771.1 hypothetical protein TcasGA2_TC031700 [Tribolium castaneum]; SEQ ID NO: 34MENFIIANNMCIFNEPNNPPTFETTNGASYIDLTIGSEFLLNKIQTWKTVDINTSDHRAITFEFNDNHNQIFPEVNASMDQKICNFNIKKAAPLLEQLSTDLINKYSILTSQNKIDQCLEKFYFELNKIISSCSRVKKTFKNRPTFWNEQIEALRRQYLKAKKDLYKNKNPDLKEALYISMSQVKQKFKDRIKLDKEKSWENFVKEDLAPNPWGVVYRVAAEKFKKTNLLGVFENDGETSLNPSDAAKRLLHSLLPDDNEENETLSQKITRQDFNTIEMTYRYNIDEVTNTELEIIIKNLKKKAPGLDKIDGNLTKIMHPSLSNFLLHIYNSCLRASYFPKCWKMGNLVVIPKDSGGDPSDIKNYRPITLLNDLGKIFEKLIRTRLFKSTDVFHADNQYGFCVGKSSTDALLFFKNQLQAYNQKYKYTAAVFFDISGAFDNVWYPSIIKSLRQKGVSPHLIRIMKSYVSDRNVIYSYRGIVNRKNCTKGCPQGSVLGPTLWNTILDEFLRKKIAPNTETIAYADDIVVITGANTRNEFRSTIQTIVTAVCQFAGNQKLQVSSSKTKIMMFNSPKRVHNRDLAIKINNSTIQIVTEHKYLGILFDSKFNFQKHINNVCSKARTIMMALRRKIKLRWNISVADSISAIYNHAIIPIVTYGSEVWADRLNISKIKSKLTSLSGLASRCIAGCYASVSNDAAHVLAGVPPLDLEAARINCCKLLKKDQNCNLLGFEIEKSDFDTYKHAREYVAILVEDLWQERWDSSTKGRTTYRFRPIVQSGLRYSHSFAATQILTGHGNFMSHLQRVGKSETDECAECGVRDDPIHRLLVCPLFDVPRRDIYRSVNSPFLSLNWIIHLKSELLEPFTALPQSLFIARTSRIAPLRSDYIEHRESESGSENCFVLVQTVFQGKSRIEQHLDLDADQLISEIQKQQDLPESLPKNFVKTAFKVGNKDGKANQWVVELHPVARNHFIKSGSRLFINWKSLHIRDYLRVTRCFKCQKFGHVSKFCNSEKQCGYCASTDHESVTCKLKNEENKHKCIAAVFGSSPRTLTIPGEGHVEASRRFVPISAAGLQGEEPLVDRIMIGSEDRGVSGLARKRVAADVPGLCEVKRCSVSAVNVESEFGPVPRPPAEPSCCEAPGAPVRARAMGLTTLSGTKTSNSGAQGPSTSAPMQNMAGGFVCDCGRSYALKTSLARHKKECGKNNTECRWCGTRFNTLAGTRQHERKAHFVQYQSDLAKALPQPESELMEKIAIVEARSSNGIFYKEMMASTGLTHQQVRSRREKPEYKGFLERARRSLAQTNIRAGSISPASTIAGSLESASPKAGCSSSASPGPTTRSRAPTKGVPLRSSNSARIVVEAQVHTRAPPNTGETEVALRESRRTVPRLGPNPSRPCGISPLMAIAIDEDSVLGGLRVQAEPSPTAVHSVETFPGTSSMTPMETDRVHNKSGIDPILEHNGTRQVRREESSTREDPVEQWSPNYPKTPVTMPNITTTADASCTSYNRTPQTLPGNRRRRSRSLPPVQRKSASDDLESVDSLGPWAVFLQDQVDAGSLSGNDSLADLVRVALTKSDRGVLNDAVNRYLAQRAESLRIRKRGSKGKRKSKTGRHYGQTTSGSGQRAALFKKHQDLFLKNRRGLAETILSGKEDFGPRPEPPVTSVEEFYGGIFESPSPPDNEPLEVRATGVEDPPTYITMDEIKAARAGWQISAPGSDQIPVAAVKTMSELELAILFNIILFRNVQPSAWGVLRTTLVPKDGDLRNPANWRPITISSAMQRLLHRVLAARLSKLVSLSSSQRGFTEIDGTLANALILHEYLQYRRQTGRTYQVVSLDVRKAFDTVSHCSVSRALGRFGIPSVIREYILATFGAQTTIKCGTVTTRPIRMLRGVRQGDPLSPVLFNLVMDELLEKVNEKYEGGSLQSGERCAIMAFADDLILIADRDQDVPAMLDDVSTFLERRGMSVNPAKCRALIAGAVSGRSVVRTGSSYKIHNTPIPNVDALDAFKYLGLEFGHKGVERPTIHNLSVWLNNLRRAPLKPDQKCLFIRQYVIPRLLYGMQNPQVTSKVLREADRLIRRHLKTYYHLNVHTPDSLIHASVRDGGLGIMELRKAIPRIFLGRLVKLLNKNNDSVLSSVLQSDRVRTLMGKLSTMAGEVPESTFWRNRIASGPLSKGLEQAAEDSASRLWISEKPSGWSGRDHVRAVQLRTGNLPTKAIPSVPVGQRRCRHGCACDESISHVLQMCPLTHADRIRRHDEVVKKVARHCTSRGWTVEVEPHIRSRCGRLFKPDLAVHQPGGAIVIADVQISWDSESLTVPYERKRAKYDVPQFHQAAQHAWPGKALTFAPVIVGARGIWPRINNDRSAALQIPPVVRRACVNSVVKWGSSIHATFMSETTAKGTGLEKLAGKEDPVELDSSLALXP_002412745.1 reverse transcriptase, putative [Ixodes scapularis]; SEQ ID NO: 35MACSPHQFHPQKEPAALPSDFRPITVGPVLQRLFHKILAKRVMAAAPLDFRQRAFQPVDGCAENILLLSTVLDEARCRLRPLHLASVDLAKAFDRVTTEAILRGALRAGFDDAFLAYLRELYATSYTTLQYGGEELVVQPTTGVRQGDPLSPVLFNMVLDEFLSSVDPRVAFRSGDFTVDAMAFADDLVVCASTPQGLQQRLNDLAAFLSPRGLNINVAKSFTLSLQPSAREKKCKIVTTNRFHINGEPLPVSGVASVWRYLGVSFTPDGTRSNGVEMELEELLERVKKAPLKPQQRLLVLRTYLLPRLFHRLILGPWSVGLLKKLDTKVRAALRSWLALPHDVPLGYFHAPVGEGGLGVVSLRASIPSMRLRRIEGLRFSDHPGCAEALRCPLLLEGFPFRNKNBAE46603.1 reverse transcriptase, partial [Eptatretus burgeri]; SEQ ID NO: 36ALAFADDLVLCARTSSGLRRSMDAVQTRLSAAGLVLNVEKSATLAITIDAKAKRYVVDTSEVFYLGQQNLGVLDAASQLKYLGIQFLPRGAAPSDGSALEKGLRNLRRAPIKPQQRMFMLRDHLIPQLQHGLVLGAARRGTLRKLDVQMRHEITRLWLRLPKDTPVAYFHARSADGGLGLPQFSVTIPILRERRMRGLEQSDSPYVRAVIRTKLGDVVRSRNTYNLHYGGERPRSIKQADVITAKLLHQAVDGRGLLEASRVPEPNDWVLGRSRLQSGRAFIDSVKVRGNLLPTGSRSSRGRRGQGSEGWCDAGCRAKESLNHISQACIRTHGGTVQRHDAVSRFACGRLRQRGFVVIEEPRIPTPAGIRKPDHAEKNGKPVILDTQIKSDTLKLSDEHVRKRNYYDTPEVLDWVRNKFESENVPLVSTITLSWRGVWAPESATLLRELGLTKTDLRLISVMVVEKTALMFRWFKRSALTRNAPRQAF020000.1 R2 protein, partial [triops cancriformis]; SEQ ID NO: 37PIIFNLVINGAIRRLPDEFGFEVSPKIFLNCLAYADDLILVATTRIGLKRLIIIVGEYLQRRGLKLSAEKSIGLSITACGKEGLTYVSSANKIDFQGVEIKNMCVLDTWQYLGIQFSHIGCTDRITPEVSDLLFKLQKAPLKVQQKLYALRHYLIPRLLHGLILSRVLITELKSIDNLIRKYVRSLLHLPKDTPLGYLYASTKDGGLGIPCLRYLILKCRLARILRMRSSNDAVVREIANSEFLLHETQRLRDSLSIDGSVLDTNELIRKYWAKRLYTSYDGKSLAYSEFFPYGNSWVREDHMNQRAHVFCDCIKLRINALPTRARTSRGRTESKDSSCRAGCKFSSGIPMRETVNHITGVCQRTHDARVRRHDSIVDFLVSIWKTKSTNEVYKEPNYRTPLGLRKPDIVVKTPNEVWIADVQVLADNANLEKEYSIKTNKYASDAGLIQGLKKQFPNINLFSFFAVTINNRGLISRSSVNELRKRGVNSRDMLTIILRAMEGSLMIWRIFNQTTSSARKRX36111.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Trichinella murrelli]; SEQ ID NO: 38MGKRSTDNVNKLEGPLSNNEAPVNNRMVTRSTIKRATVVDSRSAPPLLVRPPVESPEFKLVCATIDNRRQKSTASSAPLNNGGICQPLSEAPDDNAAVVSNSCAVSEIKEMPPARINMRLRSRKACGNIPEPPRLPRTSQTASESHDGSQPCPGSSTAITSFPRASDNHPVSTNTQGTHVCSVCSRVFNSFPGLRLHEKRAHPATFAASSQKSVKHLWTTDHLREVKEVEEILAANNSRSVKALAEALSRKWSEDISMDMAKYLRKKLRAVDLNSAQPTNTALTDGVNSPREGPENLTEEGESLNNGSQVIGTEIMGPERFPECDSFQTHSANADLSNPCPQHPDGLLSEQGLDRDQGGVLRKHLEDGLASCNSELEGLLNFIVSKVLNSGIEDRNKHVDAAISVLTEFLCESKSHQPPPPRKKRTREEPKNRRQKIRSKYAQMQTLFKRDPKRVAAHLIRNQPLCNVSCPIDAAESALRQRLSQRPGVDAAPITSKCPQNSKNILDPIFPEEVTLHLQKMKIHTSAGPDGIKVSHLRSCDPVCLAKAFNLFLLARHIPQQLKDCRTTLIPKTDDPRPDAEDYRPITVASCLYRLFSKIVTRRLEDSLSLHPRQKAFRSGTDGAFDNTSTLMTVIREAHNCGKELNIVSIDLAKAFDTVNHTSITRALRMHGLDDESRTLITEMVTGSSTIIKGDGGALSNRIEINQGVRQGDPISPLLFNAVMDELVERLERTGEGFKLKGVEVTTLAFADDVTLISRSHRGMEKLLSITLDFLNERGLQLNINKCKGIRLVRTPKTKSLVEDTSKPFRVPSFGEENQHIPMVLPGDLIKFLGIDITLNGKPHFDLAPLEDTLERIRKAPLKPAQKLATVRDYLIPSLEYRLGVPGISRKLLESVDGAIRLTVKRFLHLPLTGMNSMFLSMPVKEGGLGLRSLSTQHIARLAVGTNSMSISTDTVSRVVADTTTLRKPLLSALEHFAVPTATKSAIREGKRNLLRAEIAQLSETYQGSCLPSFKHGSLVNTWLRGTSGMRSRDYITGLKLRFGVIETRSQKWRGRTPQNPDALLCRHCGHSSGNRETAAHVSQKCLVTHALIVQRHNKIVRLVGDRAKDEGFAVHVETAVKSGEEVYKPDLILIKADTAHIIDVAVPWEKGTNMHEKHERKTNKYAQLVDDVKALFGVQNCTVGALVIGARSSWCTSNDGSLKACGLHLPKKTDGEDLTTEADDSDAEPWQKPEHSPPHAKENTEDRNTEEQSEPYTTPQTLRTSENPEIQRRRRLHRTTTRRDCARRTDHNWTPERGTTHPQKQGPKRZ66264.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Trichinella papuae]; SEQ ID NO: 39MGKPSRPAQETPSTAAGSDGQARSGLRRRNHAEVTYPGPFTCTVCSLTECVYSRFKNHCMTHHLHLELSCSVCRKVFPTINAVACHYPHCAKTPRNPNISQNIPETTQVGRTVASSINNKGISENKEPPQNNRVVTRSQVKRAAVGAVTRSRSSLCNGAPAVNAAAGIVRSQVNVVFPDCATLNNNSRQRLMVAPAPPNNGELCSPLSGKVTDGGNVIVRRSARLASNCEAGLHNNGADASSVANVPVIKEVPPARTYRRNRPASANTNITETPRQPPTSMTVPECRRRTREQPQRTTASEATQPAAPTVADNAFPCGECGRVFSTFAGMRLHLKRAHPSSFSSLQPPVKVPRWSALESDTLRELEDALRRNGELSNEKLASLMTDRFERVFTIDMVKGHRRKFRETPRTEGENSRPSTPRESTPAPTAQTSPSTMPVNNITRDNPETAEDINKKLKQHLLHCTTTHNTEVEAEINDIIRNHIIKGNNKNYVTRIVKYLIGAMRPEEERGPKKGRKKKKPEPSVPLNSKQRKRMAYRKVQQAYHKDPKRVVAHLFHSQPLENVSCPVESGEKALQARLGKRPPADRAPFLPKRAPLKNHLLSPISAKEVSEHLKQMNLASASGPDGVKVSHLRDIGPQCLSKIFNTFLLERHIPQVLKDCRTTLIPKVDNPRPDAEDFRPITIGSCIYRLFSKIVTSRLSQLTPLNPRQKAFRSGTDGAFDNITTVASLLKLARKTGKEINLACIDLAKAFDTVNHTSITRALHRHGVDSASIELVESMVGEATTVIINSDGTRSNVIKFNRGVRQGDPISPLLFNLVLDELIDNLDQARCGFSITKEIQVSCVAFADDITLVSGSREGMNNLLTITREFLGERGLGINHSKCKGIRFTKVPKSKSLIIDTNPNCFLIRNQQGTPEPIPMAKPGEPLKTLGINLTLEGNPTFNYPELTRILNTIKHAPLKPHQKVQIIRDHLIPLLQYKLGVPTFYRATLNNIDKSIRLTVKEILHLPTTGLHNSYLYLPLKEGGLGLKRLATQYASRVGLGLSNMATSDDAVSRAVAGLHLSLMDKAKNCLGLSEISKEAIKKAKEKLVQAEIRTLLQCHLGRSHSSFTNDTISNSWMRYPTFLSARNYIMGIKLRAGIIEIRAQKWRGRSPPHPTMLLCRHCGARSRTRETDIHVSQKCLHNKKLILRRHNCVVSTLGRRATQQGFAVYYEPCIKHGETVLKPDLVIIKGDTATIIDVAVPWEQGTNLREHNSRKISKYQCLEREAAKYFNVKTVKTGSLVVGARGKWSAGNDSTLKSCGLHCSKRLKKLLCTIALEGTCAVFKHKRY45664.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Trichinella britovi]; SEQ ID NO: 40MVTRSTIKRATVVDSRSAPPLLDRPPVERPEIKLVCATIDNRRQKSTAASAPLNIVGICQPLFEAPDDNAAVVSNNCAVSEIKEMPPARINMRLRSRKACGNILEPPRPPRTSQSASESHDGSQPCPGSSAASTNSPRASISQPASTNAQGTHVCSVCSRAFNSFPGLRLHEKRAHPATFAASSQKSVKHLWTIDHLREVKEVEEMLAANNSRSVKALAEALSRKWSEDISMDMAKYLRKKLRAVDLNSAQPTNTALTDGVNSPREGPENTTEEGESLNNGSQVVGTEIMGPERLPECDSLQTHSANADLSNPCPQHPDGLLSEQGLDRDQWGVLRKHLEDGLASCNSELEGLLNFIINKILNSGSEDRNKLVDAAISVLIEFLGEPKSHQPPPPRKKRTREEPKNRRQKIRSKYAQMQTLFKRDPKRVAAHLIKNQPLCNVSCPIDAAESALRQRLSQRPGVDAAPFTSKCPQNSKNILDPIFPEEVTLHLQNMKLQTSAGPDGIKVSHLRSCDPVCLAKAFNLFLLARHIPQQLKDCRTSLIPKTDDPRPDAEDYRPITVASCLYRLFSKIVTRRLEDSLSLHPRQKAFRSGTDGAFDNTSTLMTVIREAHSCGKELNIVSIDLAKAFDTVNHTSITRALRMHGLDDESRTLITEMVTGSSTIIKGDGGALSNRIEINQGVRQGDPISPLLFNAVMDELVERLEQTGEGFKLKGVEVTTLAFADDVTLISRSHRGMEKLLSTTLDFLNERGLKLNISKCKGIRLVRTPKTKSLVEDTSKPFRVPSFGEENQHIPMVLPGDLIKFLGIDITLNGKPHFDLAPLEATLERIRKAPLKPAQKLATVRDYLIPSLEYRLGVPGISRKLLESVDGAIRLTVKRFLHLPLTGMNSMFLSMPVKEGGLGLRSLSTQHIARLAVGTNSMSISTDTVSRVVADTTTLRKPLLSALEHFAVPTATKSAIREGKRNLLRAEIAQLSETYQGSCLPSFKHGSLVNSWLRGTSGMRSRDYITGLKLRFGVIETRSQKWRGRTPQNPDALLCRHCGHSSGNRETAAHVSQKCLVTHALIVQRHNKIMRLVGDRAKDEGFAVHVETAIKSGEEVYKPDLILIKDDTAHILDVAVPWEKGTNMHEKHERKTNKYAQLVGDVKALFGVQNCTVGALVIGARSSWCPSNDGSLKACGLHLPKKPDGEDLKTEDNHPCAESRQRPKHSPPLEEENTEERNTAEQTEPCTTPQTLRTTENPDIRRRTRLNRTTTRRDCARRTGFINWTPYIKTDLCRNIGLNLSLIKSHWCASQIRLTAAGTFLRESPANSATSQMPRHLISDAYEWINKIPSVPIYYLAKPQPRERAWQNQRGKKTLLSLTLVCAJ00246.1 TPA: polyprotein [Schistosoma mansoni]; SEQ ID NO: 41MPVSTGAETDITSSLPIPASSIVSPNYTLPDSSSTCLICFAIFPTHNILLSHATAIHHISCPPTPVQDGSQQMSCVLCAAAFSSNRGLTQHIRHRHISEYNELIRQRIAVQPTSRIWSPFDDASLLSIANHEAHRFPTKNDLCQHISTILTRRTAEAVKRRLLHLQWSRSPTAITTSSNNHTITDIPNTEARYIFPVDLDEHPPLSDATTPNASTHPLPELLVILTPLPSPTRLQNISESQTSHESNKNSMHTPPTYACDPDETLGATPSSTIPSCFHSYQDPLAEQRGKLLRASASLLQSSCTRIRSSSLLAFLQNESTLMDEEHVSTFLNSHAEFVFPRTWTPSRPKHPSHAPANVSRKKRRKIEYAHIQRLFFIHRPKDASNTVLDGRWRNPYVANHSMIPDFDCFWTTVFTKTNSPDSREITPIIPMTPSLIDPILPSDVTWALKEMHGTAGGIDRLTSYDLMRFGKNGLAGYLNMLLALAYLPTNLSTARVTFVPKSSSPVSPEDFRPISVAPVATRCLHKILAKRWMPLFPQERLQFAFLNRDGCFEAVNLLHSVIRHVHTRHTGASFALLDISRAFDTVSHDSIIRAAKRYGAPELLCRYLNNYYRRSTSCVNRTELHPTCGVKQGDPLSPLLFIMVLDEVLEGLDPMTHLTVDGESLNYIAYADDLVVFAPNAELLQRKLDRISILLHEAGWSVNPEKSRTLDLISGGHSKITALSQTEFTIAGMRIPPLSAADTFDYLGIKFNFKGRCPVAHIDLLNNYLTEISCAPLKPQQRMKILKDNLLPRLLYPLTLGIVHLKTLKSMDRNIHTAIRKWLRLPSDTPLAYFHSPVAAGGLGILHLSSSVPFHRRKRLETLLSSPNRLLHKLPTSPTLASYSHLSQLPVRIGHETVTSREEASNSWVRRLHSSCDGKGLLLAPLSTESHAWLRYPQSIFPSVYINAVKLRGGLLSTKVRRSRGGRVTNGLNCRGGCAHHETIHHILQHCALTHDIRCKRHNELCNLVAKKLRRQKIHFLQEPCIPLEKTYCKPDFIIIRDSIAYVLDVTVSDDGNTHASRLLKISKYGNERTVASIKRFLTSSGYIITSVRQTPVVLTFRGILDRASSQSLRRLCFSSRDLGDLCLSAIQGSIKIYNTYMRGTQ03278 Retrovirus-related Pol polyprotein from type-lretrotransposable element R2 [Nasoniavitripennis (Parasitic wasp)]; SEQ ID NO: 42NQIKKSNTSTGARIPKAMTNPADNFAGGQWKPPGRRSARTSATGMFVCEHCLRAFTTNTGRGLHIKRAHEEQANEAITTERSRARWTNEEMEAVQAEIDCEGRTAINQEILRIIPYQRTIDAIKCLRKQQKYKTIRERVANRRAENRARETELTRLETADEDPASQEQDNPNMSLKNWLKEVIESDDDRLCADLRTAIEMALAGQSPLDVCTVGCYQYTMTNLPLVPVRLGGPIYWCNAQSRSNPGETQRRQTIKESNNSWKKNMSKAAHIVLDGDTDACPAGLEGTEASGAIMRAGCPTTRHLRSRMQGEIKNLWRPISNDEIKEVEACKRTAAGPDGMTTTAWNSIDECIKSLFNMIMYHGQCPRRYLDSRTVLIPKEPGTMDPACFRPLSIASVALRHFHRILANRIGEHGLLDTRQRAFIVADGVAENTSLLSAMIKEARMKIKGLYIAILDVKKAFDSVEHRSILDALRRKKLPLEMRNYIMWVYRNSKTRLEVVKTKGRWIRPARGVRQGDPLSPLLFNCVMDAVLRRLPENTGFLMGAEKIGALVFADDLVLLAETREGLQASLSRIEAGLQEQGLEMMPRKCHTLALVPSGKEKKIKVETHKPFTVGNQEITQLGHADQWKYLGVVYNSYGPIQVKINIAGDLQRVTAAPLKPQQRMAILGMFLIPRFIHKLVLGRTSNADVRKGDKIIRKTVRGWLRLPHDTPIGYFHAPIKEGGLGIPAFESRIPELLKSRIEALGASNMQTARSLLGGDWVAERKKWINTQKIKNSEWAQKLHLTTDGKDLRDTRKAEASYSWIRDIHVAIPASVWIKYHHTRINALPTLMRMSRGRRTNGNALCRAGCGLPETLYHVVQQCPRTHGGRVLRHDKIAEQVAIFMQEKGWLVLREAHIRTSVGLRKPDHARKGQDCKIIDCQIVTTGNDIRIQHERKIQYYASNWELRRSAATMIGHQGQVSVEAITISWKGVWEPRSYCLLRDCGIPKVKIKGLTTRVLLGAYLNFNTFSKATYRTERRRTANQ03279 Retrovirus-related Pol polyprotein from type-1 retrotransposable element R2 [Bradysia coprophila (Dark-winged fungus gnat) (Sciara coprophila)]; SEQ ID NO: 43VSVPPTSYRDRIMNALEESMIDVDAIRGSSARELVEIGKVALLNQPMDEGRIMSWLSDRFPDTQKPKGPIYSKTTIYHGTNKQKRKQRYALVQSLYKKDISAAARVVLDENDKIATKIPPVRHMFDYWKDVFATGGGSAATNINRAPPAPHMETLWDPVSLIEIKSARASNEKGAGPDGVTPRSWNALDDRYKRLLYNIFVFYGRVPSPIKGSRTVFTPKIEGGPDPGVFRPLSICSVILREFNKILARRFVSCYTYDERQTAYLPIDGVCINVSMLTAIIAEAKRLRKELHIAILDLVKAFNSVYHSALIDAITEAGCPPGVVDYIADMYNNVITEMQFEGKCELASILAGVYQGDPLSGPLFTLAYEKALRALNNEGRFDIADVRVNASAYSDDGLLLAMTVIGLQHNLDKFGETLAKIGLRINSRKSKTVSLVPSGREKKMKIVSNRRLLLKASELKPLTISDLWKYLGVVYTTSGPEVAKVSMDDDLSKLTKGPLKPQQR1HLLKTFVIPKHLNRLVLSRTTATGLCKMDLLIRKYVRRWLRLPGDVPVAFLYAPVKAGGKGIPCLKQWIPLMRFLRLNKAKRTGGDRIAAVLNCQLYASISHSCKTGPVSVGLWRSTNTGGLSAYWRRILIGMVDGKDLKSAQNHSSATSFNSIRMNDISGEDYIHYNQLRTNSIPTRKRTARGRPNKPTACRAGCDKLKRLQHDIQGCIRSQGGLVQRHDRVVDLLFDECETKGYAAEKVVHLRTSEELWKPDLVLKKNGRVVVVDAQVVQCGRLESDHRVKVSKYRDDPELADVIREKYAVQEVTFEACTLSYKGIWSKNSVEGLQKLGISNYCLFKIVTSVLRGSWLNWVRFNNVTTVVH WKXZ75830.1 hypothetical protein TcasGA2_TC031908 [Tribolium castaneum]; SEQ ID NO: 44MVPLLLVLDLEPTVLTTPLDLRERTMLMDMDSEDEAGEHGPPADNAHLTSGEPIEIILMLPFQSRSCICLNAGKGNFRATADGDLESWCGSDVIAHLRKTSWRKPQTGMKSRSFRRIGDCAAGSSRRGVRLTGKAGREGRFAASPHLSPRYLAGSVSGNVPSVPPNPGLGAGAPAFAAVPNADGGPAQNPCPYCARSFTTANGRGLHIRRAHPDEANNAIDIERIHARWSHEETAMMARLEAGAIQRGGVRFMNQFLVPRMPGRTLEAVKSKRRDATDKALVQRFLQGDRLCNIARRACDGGDVSGQLLGWLRDVFPVKRVSTRGDQSNLDVDGALVSRHTARTREYARVQELYRKDPKACLARIFGDRREGANRAPNRDPAFIDFWRGVFSEASAEVEGCAEEVSDHGGGKVSGPEWCSAGARRNSGFRVEQLPPEAAALLFNVLLLGRCLPAELTRTRTVFIPKTDAPRTPADYRPISIASVVARHFHRVLSAHVQRIPDLFTKYQRGFLSGVDGIADNLSVFDTMLTMSRRCCKHLHLAALDVSKAFDTVSHFAIVRACRETHSLLSSLTWSWTGLLKRLSTDVGFRLTDATKVTALAFADDVVLCATTARGLQTNLDVLEAELRLAGLLLNPNKCQALSLVASGRDHKVKLVTKPTFTVGQNTILKGSGMCGCGSEGVAAGLKRNTCAPLKPQQRMHLLRVFFLPKFYHAWTFGRLNAGVLRRLNVVVRTSVRTWLRLPHDIPVGKFHAPTKSGGLGIPQLSRLIPFLRLKRFDRLGRSAVDYVRECAFTDIADQKIRWCRERLSGIVDQVAGGRDALDAYWTAQLHQSVDGRALRESASVASSTQWLRCSTRAIPASDWLHYTAVHIGALPSRVRTSRGRRGGQDVSCRGGCLLDETPAHCIQVCHRTHGGRMLRHDAIAKRISADLMELGWIVTREVSFRTTAGVFRPDMVAVKEGVTVILDVQIVSPAPTLDEAHRRKVAKYRDRADLARYLAEAAVARGRAPPANIRFASATISWRNVWSAESVGSLRELRLSARHFNRYTTMSFCGSWRNWVRFNASTASGMGRGRGDASPRRHENQHDNDSLADLVRVALTKSDRGVLNDAVNRYLAQRAESLRIRKRGSKGKRKSKTGRRYGQTTSGSGQRAALFKKHQDLFLKNRRGLAETILSGKEDFGPRPEPPVTSVEESYGGIFESPSSPDNKPFEVRATGVEDPPTYITMDEIKAARAGWQISAPGSDQIPVAAVKTMSELELAILFNIILFRNVQPSAWPSQGAVRPCRWWEGLSTCNESAGNAGGSHGRDSWQGHPCQP16423.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Drosophila melanogaster]; SEQ ID NO: 45FERKNFSDGLVPQRKFIHIGTTSTNNEPRIPLHNLMTTRPSVDIFPEDQYEPNAAATLSRVPCTVCGRSFNSKRGLGVHMRSRHPDELDEERRRVDIKARWSDEEKWMMARKEVELTANGCKHINKQLAVYFANRSVEAIKKLRQRGDYKEKIEQIRGQSALAPEVANLTIRRRPSRSEQDHQVTTSETTPITPFEQSNREILRTLRGYSPVECHSKWRAQELQTIIDRAHLEGKETTLQCLSLYLLGIFPAQGVRHTLTRPPRRPRNRRESRRQQYAVVQRNWDKHKGRCIKSLLNGTDESVMPSQEIMVPYWREVMTQPSPSSCSGEVIQMDHSLERVWSAITEQDLRASRVSLSSSPGPDGITPKSAREVPSGIMLRIMNLILWCGNLPHSIRLARTVFIPKTVTAKRPQDFRPISVPSVLVRQLNAILATRLNSSINWDPRQRGFLPTDGCADNATIVDLVLRHSHKHFRSCYIANLDVSKAFDSLSHASIYDTLRAYGAPKGFVDYVQNTYEGGGTSLNGDGWSSEEFVPARGVKQGDPLSPILFNLVMDRLLRTLPSEIGAKVGNAITNAAAFADDLVLFAETRMGLQVLLDKTLDFLSIVGLKLNADKCFTVGIKGQPKQKCTVLEAQSFYVGSSEIPSLKRTDEWKYLGINFTATGRVRCNPAEDIGPKLQRLTKAPLKPQQRLFALRTVLIPQLYHKLALGSVAIGVLRKTDKLIRYYVRRWLNLPLDVPIAFVHAPPKSGGLGIPSLRWVAPMLRLRRLSNIKWPHLTQNEVASSFLEAEKQRARDRLLAEQNELLSRPAIEKYWANKLYLSVDGSGLREGGHYGPQHGWVSQPTRLLTGKEYMDGIRLRINALPTKSRTTRGRHELERQCRAGCDAPETTNHIMQKCYRSHGRRVARHNCVVNRIKRGLEERGCVVIVEPSLQCESGLNKPDLVALRQNHIDVIDTQIVTDGHSMDDAHQRKINRYDRPDIRTELRRRFEAAGDIEFHSATLNWRGIWSGQSVKRLIAKGLLSKYDSHIISVQVMRGSLGCFKQFMYLSGFSRDWTKRX34481.1 Retrovirus-related Pol polyprotein from type-2 retrotransposable element R2DM[Trichinella murrelli]; SEQ ID NO: 46MSNRLANTAAAGGVPEKNSGTLDIPGQPSSSGEKRAISYPGPFGCNSCSFTSTTWLSMELHFKSVHNTCEFVFLCSKCEKSWPSINSVASHYPRCKGSVKAAVVPSSLTNTCTTCGSSFGTFSGLQLHRKRAHPDVFAASCSKKTKARWSNDEFTLLARLEAGLDPACKNINQVLAERLMGYNITRGVEMIKGQRRKDQYKALVRQLRSNSETQQCVGLAGSMDLNVPVNDTTSSVASEVTITYPEYGAVMSCDLIKEATGMAMVDVNELQSNIRKLFSSGRKLPMKLRGAREAVQKKMANPRVAKFKRFQRLFRSNRRKLANHIFDKASLEQFGGSIDEASDHLERFLSRPRLESDSYSVISGDKSIGVAHPILAEEVELELKASRPTAVGPDGIGLEDIKKLNSYDLASLFNLWLKAGDLPESVKASRTIFLPKSDGTTDISNCRPITIASALYRLFSKIITRRLAARLELNVRQKAFRPEMNGVFENSAILYALIKDAKVRSKEICITTLDLAKAFDTVPHSRILRALRKNNVDPESVDLISKMLTGTTYAEIKGLQGKPITIRNGVRQGDPLSPLLFSLFIDEIIGRLQACGPAYDFHGEKICILAFADDLTLVADNAAGMKILLKAACDFLEESGMSLNAEKCRTLCISRSPRSRKTFVNPAAKFNISDWKTGVSSEIPSLCATDTFRFLGHTFDGEGKIHIDTEEIRSMLKSVKSAPLKPEQKVALIRSHLLPRLQFLFSTAEVDSRKAWLIDSIIRGCVKEILHSVKAGMCTDIFYIPSRDGGMGLTSLGEFSLFSRQKALAKMAGSSDPLSKRVAEFFIERWNIARDPKVIEAARRVYQKKRYQRFFQTYQSGGWNEFSGNTIGNAWLTNGRARGRNFIMAVKFRSNTAATRAENLRGRLGMKECRFCKSAIETLAHICQKCPANHGLVIQRHNAVVTFLGEVARKEGYQVMIEPKVSTPVGALKPDLLLIKADTAFIVDVGIAWEGGRPLKLVNKMKCDKYKIAIPAILETFHVGHAETYGVILGSRGCWLKSNDKALASIGLNITRKMKEHLSWLTFENTIRIYNSFMKNEEB15300.1 reverse transcriptase, putative [Pediculus humanus corporis]; SEQ ID NO: 47MRTRQSKNQNKSCSTVDLRQLDENVNFTASDPGHSNDVSHRSPVQETTRSHRIRWTQEDLQELMWCYFYSQKFGSGSESDTFKIWRGRNPNSRKDMTSKKLAAQRRYIIKKIENDKLEEIKKNVDSSCSNVIRDNAPIITKLNESNNNNRYETDTDEQLLTDAEMKSIEERLIEEIKKVKMCPLINREPLRKIYKNKKATEVLHLIDNTLINVLEKVVDINLTTINEIIYAAGVVATDIILGPRKEARHKGIETKKSTSPIWIQRIEGKIKRIRSHISLVSEMKKNNNLKKRTIKKLDHLKRIYKLKTMEDIELTMETLKQKVLLYSQRIRRYKKREQFWRQNKLFESDPKKFYRTIREQNIQNGSSTLNVEKMADFWSNIWEKSHPLNKNSTWMNKEKEAHAWIASSTMSDIIMADLEICLKNTANWKSPGLDRVQNFWIKNFTSTHKYLLVSINKLIMGRQEMPEWITTGKTYLLPKKSGAMEPKDFRPITCLPTMYKIITAIIAEKIYGHLRKNNIFPPEQYGCRKGSYGCKEVLLINKLIMASAKQKRKNLSMAWIDYQKAFDSVPHEWITEALKIYKVDPKITAFCEKSMKNWCTQLEVQKYSSRKIFIKRGIFQGDSLSPLLFCMSLIPLSRQLNIKDQGYQLVPGGRKITHMLYMDDLKLYAKNEEELNKMLRTVQTFSSDINMKFGLEKCARINIVRGKLKQKQNIEDSEEELIKELDPGSSYKYLGIEENFGIANKEIKPRLKKEYFKRLRLILQSELNGRNKITAVGTLAVPVIEYSFGLVDWTKEEITHLDRRTRKILTMNGALHPKADVDRLYVSRKDGGRGLRQIEAAHQNAIIGMGKYIESHREDPILAQVIHAEEKTTKKGVLKRAKQIVQENKENEIMEEGQLATYNSKAQSQKKLIGKWEQKKLHGQYLKRINAEDINKKSTHNWLRRGKLKIETEAFITAAQDQALRTHNYEKVILKVRQDDKCRICQSQSETIDHLISGCPILAKHEYLERHNKICQYLHWSICREYGMDGLPKEWYNHIPSPVTTVGPCTVLYDQQIHTDRTVPANKPDIILRHNGEKWCKLIEVSVPAEKNTTAKEADKRLKYRNLEIEITRMWGTKTETIPVIVGALGAMPHSIKGNLKKIMKNLKEETIQEIALCGTAHILRKILXP_002431867.1 reverse transcriptase, putative [Pediculus humanus corporis]; SEQ ID NO: 48MRTRQSKNQNKSCSTVDLRQLDENVNFTASDPGHSNDVSHRSPVQETTRSHRIRWTQEDLQELMWCYFYSQKFGSGSESDTFKIWRGRNPNSRKDMTSKKLAAQRRYIIKTQKIENDKLEEIKKNVDSSCSNVIRDNAQIITELNESNHKSKTDTDEQLLTDAEMKSIEERLIEEIKKVKMCPLINREPLRKIYKNKKATEVLHLIDNTLINVLEKVVDINLTTINEIIYAAGVVATDIILGPRKEARHKGMETKKSTSPIWIQRIEGKIERIRLHISLVSEMKKNNNLKKRTIKKLDHLKRIYKLKTMEDIELTMETLKQKVLLYSQRIRRYKKREQFWRQNKLFESDPKKFYRTIREQNIQNGFSTLNVEKMADFWSNIWEKSHPLNKNSTWMNKEKEAHAWIASSTMSDVRMADLETCLKNTANWKSPGLDRVQNFWIKNFTSTHKYLMVSINKLIMGRQEMPEWITTGKTYLLPKKSGATEPKDFRPITCLPTMYKIITAIIAEKIYGHLRKNNIFPPEQYGCRKGSYGCKEVLLINKLIMASAKQKRKNLSMAWIDYQKAFDSVPHEWIIEALKIYKVDPNITAFCEKSMKNWCTQLEVQKYSSRKIFIKRGIFQGDSLSPLLFCMSLIPLSRQLNIKDQGYELVPGGRKITHMLYMDDLKIYAKNEEELNKMLRTVQTFSSDINMKFGLEKCARINIVRGKLKQKQNIEDSEEELIKELDPGSSYKYLGIEENFGIANKEIKPRLKKEYFKRLRLILQSELNGRNKITAVGTLAVPVIEYSFGLVDWTKEEITHLDRRTRKILTMNGALHPKADVDRLYVSRKDGGRGLRQIEAAYQNAIIGMGKYIESHREDPILAQVIHAEEKTTKKGVLKRAKQIVQENKENEIMEEGQLATYNSKAQSQKKLIGKWEQKKLHGQYLKRINAEDINKKSTHNWLRRGKLKIETEAFITAAQDQALRTHNYEKVILKVRQDDKCRICQSQSETIDHLISGCPILAKHEYLERHNKICQYLHWSICREYGMDGLPKEWYNHIPSPVTTVGPCTVLYDQQIHTDRTVPANKPDIILRHNAEKWCKLIEVSVPAEKNTTAKEADKRLKYRNLEIEITRMWGTKTETIPVIVGALGAMPNSIKGNLKKIMKNLKEETIQDIALCGTAHILRKIL

1.-30. (canceled)
 31. An R2 reverse transcription enzyme comprising anamino add sequence selected from the group consisting of SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO:55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQID NO: 65, SEQ ID NO: 66, and SEQ ID NO:
 67. 32. The R2 reversetranscription enzyme of claim 31, configured to perform saidamplification reaction at a substantially constant temperature.
 33. TheR2 reverse transcription enzyme of claim 31, configured to perform saidamplification reaction at a temperature of from about 12° C. to about42° C.
 34. The R2 reverse transcription enzyme of claim 31, having amisincorporation error rate of at most about 1 base out of 100 bases.35. The R2 reverse transcription enzyme of claim 31, having amisincorporation error rate of at most about 1 base out of 10,000 bases.36. The R2 reverse transcription enzyme of claim 31, configured togenerate said plurality of complementary deoxyribonucleic acid moleculesfrom said one or more template ribonucleic acid molecules with aspecific activity of from about 20,000 units/mg to about 140,000units/mg.
 37. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 49. 38. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 50. 39. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 51. 40. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 53. 41. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 54. 42. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 55. 43. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 56. 44. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 57. 45. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 58. 46. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 59. 47. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 60. 48. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 61. 49. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 62. 50. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 63. 51. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 64. 52. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 65. 53. The R2 reverse transcription enzyme of claim 31,comprising the amino acid sequence of SEQ ID NO:
 66. 54. The R2 reversetranscription enzyme of claim 31, comprising the amino acid sequence ofSEQ ID NO:
 67. 55. The R2 reverse transcription enzyme of claim 31,capable of template jumping independent of sequence identity betweensaid template ribonucleic acid molecule and an acceptor nucleic acidmolecule.